Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin.

We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca2+ -ionophore activation. The time-dependence of annexin V incorporation into the detergent-extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re-associated with the actin filaments when re-polymerization was induced in vitro. Using a bifunctional crosslinking reagent we have identified an 85-kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca2+], however, inclusion of an extract from thrombin-stimulated platelets lowered the [Ca2+] requirement for the binding of annexin V to F-actin to physiological levels. We also show that GST-annexin V mimics the physiological binding of annexin V to membranes, and that this GST-annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and gamma- but not beta-actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely gamma-actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to gamma-actin in an activation-dependent manner forms a strong association with the platelet cytoskeleton.     

Platelet cryopreservation using a trehalose and phosphate formulation

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

Annexin V binds to the actin-based cytoskeleton at the plasma membrane of activated platelets.

Immunocytochemical studies demonstrate that annexin V relocates to the plasma membranes of intact stimulated blood platelets. Anti-annexin V antibodies label the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed, glass-activated platelets and colocalize with actin. In addition, crosslinking experiments using detergent-solubilized membranes of activated platelets have identified an 85-kDa complex containing annexin V. The 85-kDa complex is also recognized by antibodies against actin, suggesting that annexin V interacts with actin. In addition, annexin V was found to associate with filamentous actin in the presence of millimolar Ca(2+). Annexin V was also shown by immunofluorescence microscopy to be associated with platelet cytoskeletons, colocalizing with actin in the presence of micromolar Ca(2+). These findings provide the first evidence for annexin V binding to the plasma membrane and to the actin-based cytoskeleton in activated platelets and indicate that annexin V may function in both cytoskeletal and membrane domains.     

Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.     

Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets.

Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.     

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.     

Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin

The functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin was studied by functional assessment of cofactor activity and Western blotting analyses of platelet releasates, obtained by stimulating washed suspensions of platelets with various agonists, including collagen, collagen with ADP, and the calcium ionophore A23187. Platelet factor V was released as a partially proteolyzed molecule that was bound to platelet microparticles, irrespective of the agonist used. Radiolabeled plasma factor V was not cleaved for up to 30 min following release when added to platelets prior to stimulation, suggesting that platelet factor V was stored in a partially proteolyzed form. Released platelet factor V possessed significant cofactor activity that was increased only 2-3-fold by either factor Xa or thrombin. The factor V subunits that expressed cofactor activity were isolated and found to consist of peptides of Mr = 220,000 and 150,000. Incubation of released platelet factor V with factor Xa or thrombin yielded the same cleavage pattern, in which two peptides of Mr = 105,000 and 74,000 appeared to be electrophoretically indistinguishable from thrombin-activated plasma factor V. Under the conditions of these studies, factor Xa activated platelet-released factor V 50-100 times more effectively than thrombin. This observation may be due in part to the existence of platelet factor V in a partially proteolyzed state, or its association with platelet microparticles following platelet stimulation. These data collectively suggest that platelet-released factor V may be the foremost initiator of prothrombinase complex assembly and function during the early stages of coagulation with additional cofactor activation accomplished by factor Xa.     

Association of fibrin with the platelet cytoskeleton

We have previously postulated that surface membrane proteins become specifically associated with the internal platelet cytoskeleton upon platelet activation (Tuszynski, G.P., Walsh, P.N., Piperno, J., and Koshy, A. (1982) J. Biol. Chem. 257, 4557-4563). Four lines of evidence are in support of this general hypothesis since we now show that platelet surface receptors for fibrin become specifically associated with the platelet Triton-insoluble cytoskeleton. 1) Fibrin was detected immunologically in the washed Triton-insoluble cytoskeletons of thrombin-activated platelets under conditions where fibrin polymerization and resultant precipitation was blocked with Gly-Pro-Arg-Pro, a synthetic peptide that inhibits polymerization of fibrin monomer. 2) Radiolabeled fibrin bound to thrombin-activated platelets and became associated with the cytoskeleton. 3) The amount of radiolabeled fibrin bound to thrombin-activated thrombasthenic platelets and their cytoskeletons amounted to about 20% of the fibrin bound to thrombin-activated control platelets and their cytoskeletons. 4) The association of fibrin with cytoskeletons and with the platelet surface was nearly quantitatively blocked by an antibody prepared against cytoskeletons (anti-C), an antibody against isolated membranes of Pronase-treated platelets (anti-M1), and a monoclonal antibody to the platelet surface glycoprotein complex, GPIIb-GPIII (anti-GPIII). These antibodies blocked ADP and thrombin-induced platelet aggregation as well as thrombin-induced clot retraction. Analysis of the immunoprecipitates obtained with anti-C, anti-M1, and anti-GPIII from detergent extracts of 125I-surface labeled platelets revealed that these antibodies recognized GPIIb-GPIII. These data suggest that thrombin activation of platelets results in the specific association of fibrin with the platelet cytoskeleton, that this association may be mediated by the GPIIb-GPIII complex, and that these mechanisms may play an important role in platelet aggregation and clot retraction induced by thrombin.     

Kinetics of thrombin-induced release and activation of platelet factor V

The kinetics of thrombin-induced platelet factor V activation were studied in suspension of washed human platelets. The effect of thrombin in stimulating the release reaction could be separated from its effect on factor V activation by use of a potent inhibitor of the release reaction, the prostacyclin analogue ZK 36374. When platelets were incubated with ZK 36374 prior to stimulation with thrombin, the amount of ZK 36374 required to inhibit 50% of factor Va formation was 15 pM. ZK 36374 at a final concentration of 1 nM was found to block instantaneously and completely the release of factor Va, whereas it has no effect neither on platelet factor V activation nor on the factor Va assay. By varying the time interval between the addition of thrombin (0.5 nM) and ZK 36374 to suspensions of 4.6 X 10(6) platelets/ml the rate of factor V release was found to be 12 pM factor V/min. In the absence of ZK 36374 the total amount of factor V released was 8 pM, whereas Triton X-100-treated platelets gave 13 pM factor V. It appeared that the amount of factor V that could be released was dependent on the thrombin concentration. Maximum release was obtained at 1 nM thrombin. The rate of factor V release increased in proportion to the thrombin concentration. The rate of factor V activation was found to be proportional to the thrombin concentration as well as to the amount of released factor V. When 4.6 X 10(6) platelets/ml were activated by 0.5 nM thrombin, the rates of factor V activation were found to be 0.3 pM and 1.2 pM factor Va/min at 20% and 90% completion of the release reaction. Therefore, the rate of factor V release was at least one order of magnitude faster than the rate of factor V activation. The kinetics of thrombin-induced platelet factor V activation were compared to those of plasma factor V activation in platelet-rich and platelet-free plasma. The results clearly demonstrate that platelets have no effect on the rate of factor V activation and that the kinetics of plasma factor V activation are identical to those of platelet factor V activation.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent- insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha- actinin, or vinculin in the complex. We found that the cytoskeleton- associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha- granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha- granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.     

Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation

To assess the possibility that hydrolysis of the platelet surface thrombin substrate, glycoprotein V, is a necessary step in thrombin-induced platelet activation, thrombin-catalyzed hydrolysis of glycoprotein V was correlated with thrombin-induced platelet activation. Hydrolysis of tritium-labeled glycoprotein V on washed human platelets was measured by the appearance of a labeled supernatant fragment, and platelet activation was measured as secretion of ATP. Hydrolysis of glycoprotein V was linear with respect to both thrombin concentration and time of incubation. The extent of platelet activation was correlated with the rate of hydrolysis but not with the amount hydrolyzed. Maximum platelet activation could be obtained with thrombin treatments resulting in hydrolysis of as little as 4% of glycoprotein V per min. Glycoprotein V was partially removed from platelets by pretreatment with either platelet calcium-dependent protease or chymotrypsin. The rate of thrombin-catalyzed hydrolysis of the remaining glycoprotein V from these pretreated platelets was as little as 1.5% the rate from control platelets, but there was no impairment of the extent of platelet activation. Thus, these protease-pretreated platelets compared with control platelets showed a different correlation of glycoprotein V hydrolysis with platelet activation. Glycoprotein V was also partially removed by pretreatment of prostacyclin-inhibited platelets with thrombin. After removal of thrombin and prostacyclin, these platelets were desensitized to subsequent activation by thrombin. Incubation of desensitized platelets with nonsaturating levels of thrombin led to less than 25% of the activation seen with control platelets but to a slightly greater hydrolysis of glycoprotein V. Thus, the desensitization to thrombin was not due to loss of ability of the activating thrombin to hydrolyze glycoprotein V. These results do not exclude a role for glycoprotein V as a component of the platelet thrombin receptor, but they indicate that there is no simple relationship between thrombin-induced hydrolysis of glycoprotein V and platelet activation.     

Association with actin mediates the EGTA-resistant binding of cytosolic phospholipase A2-alpha to the plasma membrane of activated platelets

The association of cytosolic phospholipase A₂-α (cPLA₂α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A₂ in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA₂α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA₂α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA₂α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA₂α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA₂α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA₂α with its membrane substrate via interaction with actin.     

Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets

The individual phospholipid concentrations, and their respective fatty acid distributions, in whole platelet lysates and plasma membranes derived from unstimulated and thrombin-stimulated intact human platelets were studied. This was of interest, since previous work had led to the suggestion that altered phospholipid concentrations in plasma membranes of intact stimulated cells may be of importance in mediating cellular responses. The concentrations (nmol/mg protein) of phosphatidylinositol in whole platelet lysates and plasma membranes derived from thrombin-activated platelets decreased by 37 and 45%, respectively, a compared to their corresponding controls. As well, concentrations of plasma membrane phosphatidylcholine and phosphatidylethanolamine in thrombin-stimulated platelets decreased by 20 and 9%, respectively, when compared with their control values. The amounts of phosphatidylserine and sphingomyelin in whole platelet lysates and plasma membranes were unchanged by exposure to thrombin. Fatty acid analyses revealed that thrombin stimulation of intact human platelets induced a decrease in the arachidonate content (from 37.7 to 33.1 wt.% of total fatty acid) of plasma membrane phosphatidylinositol. Similar shifts in the wt% of arachidonic acid in plasma membrane phosphatidylcholine were found. These results indicate that thrombin stimulation of intact human platelets produces a significant decrease in the mass of phosphatidylinositol in plasma membranes and raises the suggestion that the preferential depletion of the plasma membrane in arachidonoyl-containing phosphatidylinositol may be of importance in mediating cellular responses to external stimuli.     

Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the rate-limiting step in the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a signaling phospholipid that contributes to actin dynamics. We have shown in transfected tissue culture cells that PIP5K translocates from the cytosol to the plasma membrane following agonist-induced stimulation of Rho family GTPases. Nonetheless, it is unclear whether Rho GTPases induce PIP5K relocalization in platelets. We used PIP5K isoform-specific immunoblotting and lipid kinase assays to examine the intracellular localization of PIP5K in resting and activated platelets. Using differential centrifugation to separate the membrane skeleton, actin filaments and associated proteins, and cytoplasmic fractions, we found that PIP5K isoforms were translocated from cytosol to actin-rich fractions following stimulation of the thrombin receptor. PIP5K translocation was detectable within 30 s of stimulation and was complete by 2-5 min. This agonist-induced relocalization and activation of PIP5K was inhibited by 8-(4-parachlorophenylthio)-cAMP, a cAMP analogue that inhibits Rho and Rac. In contrast, 8-(4-parachlorophenylthio)-cGMP, a cGMP analogue that inhibits Rac but not Rho, did not affect PIP5K translocation and activation. This suggests that Rho GTPase may be an essential regulator of PIP5K in platelets. Consistent with this hypothesis, we found that C3 exotoxin (a Rho-specific inhibitor) and HA1077 (an inhibitor of the Rho effector, Rho-kinase) also eliminated PIP5K activation and trafficking into the membrane cytoskeleton. Thus, these data indicate that Rho GTPase and its effector Rho-kinase have an intimate relationship with the trafficking and activation of platelet PIP5K. Moreover, these data suggest that relocalization of platelet PIP5K following agonist stimulation may play an important role in regulating the assembly of the platelet cytoskeleton.     

Plasma membrane Ca(2+)-ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting platelets. During platelet activation PMCA is phosphorylated transiently on tyrosine residues resulting in inhibition of the pump that enhances elevation of Ca(2+). Tyrosine phosphorylation of many proteins during platelet activation results in their association with the cytoskeleton. Consequently, in the present study we asked if PMCA interacts with the platelet cytoskeleton. We observed that very little PMCA is associated with the cytoskeleton in resting platelets but that approximately 80% of total PMCA (PMCA1b + PMCA4b) is redistributed to the cytoskeleton upon activation with thrombin. Tyrosine phosphorylation of PMCA during activation was not associated with the redistribution because tyrosine-phosphorylated PMCA was not translocated specifically to the cytoskeleton. Because PMCA b-splice isoforms have C-terminal PSD-95/Dlg/ZO-1 homology domain (PDZ)-binding domains, a C-terminal peptide was used to disrupt potential PDZ domain interactions. Activation of saponin-permeabilized platelets in the presence of the peptide led to a significant decrease of PMCA in the cytoskeleton. PMCA associated with the cytoskeleton retained Ca(2+)-ATPase activity. These results suggest that during activation active PMCA is recruited to the cytoskeleton by interaction with PDZ domains and that this association provides a microenvironment with a reduced Ca(2+) concentration.     

Phosphatidylinositol 4,5-bisphosphate regulates activation-induced platelet microparticle formation

While the role of the cytoskeleton in microparticle formation is well-described, the role of membrane phospholipids in regulating this process is poorly defined. PIP(2) binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP(2) effects microparticle formation, PIP(2) was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP(2) into platelet membranes inhibited activation-induced microparticle formation by >or=90%. Inhibition was dose-dependent with an IC(50) of 12-18 microM. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP(2) levels on microparticle formation. Infusion of type IIbeta PIP kinase into permeabilized platelets inhibited microparticle formation by 75 +/- 8%. In contrast, incubation of permeabilized platelets with PI-specific phospholipase C augmented microparticle formation by greater than 3-fold. Evaluation of PIP kinases following platelet activation demonstrated that they were lost from platelets in a calpain-dependent manner during microparticle formation. Purified mu-calpain cleaved recombinant type IIbeta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP(2) levels, while exposure to calpeptin increased PIP(2) levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP(2) formation following platelet activation and that PIP(2) content is an important determinant of platelet microparticle formation.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.