Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Redistribution of the fibrinogen receptor of human platelets after surface activation

We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet increased rapidly during the shape changes associated with surface activation until 7,600 +/- 500 labels were present at saturation. Maximal binding of fibrinogen was followed by receptor redistribution. During the early stages of spreading, fibrinogen labels were uniformly distributed over the entire platelet surface, including pseudopodia, but the labels become progressively centralized as the spreading process continued. In well spread platelets, labels were found over the central regions, whereas peripheral areas were cleared of receptors. Receptor redistribution during spreading was accompanied by cytoskeletal reorganization such that a direct correlation was seen between the development of specific ultrastructural zones and the distribution of surface receptor sites suggesting a link between the surface receptors and the cytoskeleton. The association of fibrinogen receptors with contractile elements of the cytoskeleton, which permits coordinated receptor centralization, is important to the understanding of the role of fibrinogen in normal platelet aggregation and clot retraction.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation

Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

Identification of the fibrinogen receptor on human platelets by photoaffinity labeling

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. In order to identify the platelet fibrinogen receptor, we modified fibrinogen with the photoreactive, heterobifunctional cross-linking reagent methyl 4-azidobenzoimidate (MABI). MABI-fibrinogen was fully clottable and able to support platelet aggregation. To photoaffinity label the fibrinogen receptor, gel-filtered human platelets were incubated at 37 degrees C in the dark with 200 micrograms/ml of MABI-fibrinogen, 10 microM ADP, and 0.5 mM calcium. Irradiation of these platelets with ultraviolet light resulted in the incorporation of MABI-fibrinogen into the platelet surface. Incorporation could be prevented by excess native fibrinogen suggesting that MABI-fibrinogen had interacted with the fibrinogen receptor before photolysis. Examination of the irradiated platelets by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the photoactivated MABI-fibrinogen had been incorporated into a 105,000 molecular weight membrane polypeptide that also contained the PlA1 antigen. Thus, this polypeptide has the characteristics of the membrane glycoprotein IIIa. Previous studies have shown that thrombasthenic platelets lack this glycoprotein and fail to bind fibrinogen after stimulation by ADP. Consequently, our data suggest that glycoprotein IIIa constitutes at least one component of the platelet fibrinogen receptor.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

An antiplatelet peptide, gabonin, from Bitis gabonica snake venom.

Interaction of fibrinogen with its receptors (glycoprotein IIb/IIIa complex) on platelet membranes leads to platelet aggregation. By means of gel filtration, CM-Sephadex C-50, and reverse-phase HPLC, an antiplatelet peptide, gabonin, was purified from the venom of Bitis gabonica. The purified protein migrates as a 21,100-Da polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and as a 11,000-Da peptide in the presence of beta-mercaptoethanol, indicating that gabonin is a disulfide-linked dimer. It is a polypeptide consisting of about 84 amino acid residues, rich in Asp, Pro, and half-cystine. Gabonin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, U46619, or thrombin in preparations of platelet-rich plasma and platelet suspension (IC50 = 340-1600 nM). It also blocked platelet aggregation of whole blood. However, it apparently did not affect the initial shape change and only slightly reduced ATP release caused by aggregation agonists. Gabonin did not inhibit the rise of cytosolic calcium in Quin-2-loaded platelets stimulated by thrombin. In addition, gabonin dose-dependently inhibited fibrinogen-induced aggregation of elastase-treated platelets. In conclusion, gabonin inhibits platelet aggregation mainly through the blockade of fibrinogen binding toward fibrinogen receptors of the activated platelets.     

Bovine fibrinogen-induced 14C-serotonin and other compounds release from human blood platelets.

Bovine fibrinogen can aggregate human blood platelets by a direct mechanism (first wave) and also indirectly by releasing endogenous ADP (second wave). A primarily non-altered surface structure of the thrombocytes is important for the induction of the first wave. Under certain conditions a second aggregation wave, concomitant with the release and the availability reaction is elicited. Besides the cell's contact, the presence of ADP and calcium ions are necessary for the induction of the release reaction in human platelet rich plasma. The changes induced by bovine fibrinogen thus follows the same pattern as the irreversible phase of aggregation, linked to release as induced by ADP.     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.     

Daily Variations of Functional Parameters and Density Distribution in Human Blood Platelets

Blood platelets play a critical role in the onset of myocardial infarction, which has been shown to have a circadian rhythmicity with a peak incidence in the morning. In an attempt to correlate platelet parameters with the outcome of cardiovascular diseases, we studied the daily (24-h) variation of the following platelet parameters: distribution pattern of functional heterogeneous platelet subpopulations; serotonin uptake; ketanserin binding; aggregation upon thrombin, serotonin, and ADP stimulation; and platelet count. Furthermore, we analyzed the tryptophan and serotonin concentrations in the blood samples. The percentage of less dense platelets, which represent the subpopulation with the highest preactivation, showed a rhythmicity period of 24 h and an acrophase at 21:18 h. The time course of intermediate and high density platelets was inverse to that of low density platelets. The serotonin uptake exhibited also a rhythmicity with a 24-h period. The acrophase was at 13:50 h. The aggregation curves were inverse to the ketanserin binding curves. The serotonin concentration exhibited a 12-h rhythmicity. The results obtained suggest that (a) changes in platelet activity are reflected by several parameters of platelet function that underlie daily variations; (b) the aggregation curves show a peak in the morning, with an additional peak in the afternoon; and (c) changes in the distribution pattern occur independently from variations in platelet functions like aggregation and serotonin binding.     

Hypergravity results in human platelet hyperactivity

Thrombotic diseases or fatalities have been reported to occasionally occur under conditions of hypergravity although the mechanism is still unclear. To investigate the effect of hypergravity on platelets that are the primary players in thrombus formation, platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for 15 minutes. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was significantly increased. The number of platelets adherent to immobilized fibrinogen and the area of platelets spreading on von Willbrand factor (VWF) matrix were increased simultaneously. Flow cytometry assay indicated that integrin αIIbß3 was partially activated in 8 Gexposed platelets, but there was no significant difference in P-selectin surface expression between platelets treated with 8 G and 1 G control. The results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events, suggesting a novel mechanism for thrombotic diseases occurring under hypergravitional conditions.     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

The molecular nature and consequences of lipoprotein (a)'s association with platelets

Lipoprotein (a) (Lp (a)) may be pro-thrombotic in humans due to its apolipoprotein (a) (apo(a))-mediated decreases in fibrinolysis. Such decreased fibrinolysis arises putatively from interference with plasminogen conversion to plasmin due to the considerable homology between apolipoprotein (a) and plasminogen. However, in vitro, most studies have shown that human Lp (a) decreases agonist-stimulated platelet aggregation while in vivo it appears to decrease aggregation as implied by increased bleeding times with higher blood serum concentrations of Lp(a). Lp (a) binding to platelets mediated by apo (a) increases platelet intracellular c-AMP levels in resting platelets, and decreases platelet production of thromboxane A2 and fibrinogen binding to platelets all of which reduce platelet aggregation. One, though not the only, explanation of these conflicting data may be that Lp(a) self-regulates its interference with fibrinolysis by reducing platelet aggregation and platelet binding of fibrinogen and hence the degree of requirement for fibrinolysis. However, it is concluded more in vivo work needs to be done to fully understand whether, if at all, Lp(a) in varying concentrations and isoforms, favours reduced platelet aggregation or fibrinolysis.     

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.     

A study of the kinetics and mechanism of ADP-triggered platelet aggregation

The time-course of ADP-triggered aggregation of human blood platelets has been followed by sensitive right-angle light scattering intensity measurements as a function of the platelet and fibrinogen concentrations. Rayleigh-Gans light scattering theory has been combined with the Smoluchowski aggregation model to predict the dependence of the right-angle scattering intensity on particle size and concentration as well as the time-dependent changes during aggregation. The validity of the calculations was confirmed by measuring the scattering intensity with suspensions of polystyrene microspheres of known radius, as well as the time-dependent changes in the 90 degrees scattering intensity during aggregation of these particles. However, in contrast to the predictions of the model, the time-course of the scattering intensity changes during platelet aggregation was characterized by single exponential decay with a rate constant which reached a limiting value of 0.017 s-1 at high platelet concentrations. The value of kagg was also independent of the fibrinogen concentration over a 30-fold range. Covalently cross-linked fibrinogen dimers and Fragment D-inhibited fibrin protofibrils yielded aggregation rates that agreed with those measured with fibrinogen. The results indicate that the rate of platelet aggregation is not limited by either the rate of fibrinogen binding or the frequency of platelet-platelet collisions under these conditions.     

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.     

Effects of prolonged administration of high molecular levan on hematological parameters in rabbits

Rabbits were injected intraperitoneally with 100 mg of high molecular levan, daily for 4--6 weeks. Study of various hematological parameters revealed that the treatment resulted in the following changes: 1. Marked elevation in fibrinogen levels and in the number of platelets. 2. Insignificant changes in the intrinsic and extrinsic clotting mechanisms and in platelet aggregation. 3. Some reduction in the number of circulating RBCs. The findings indicate that levan treatment has no obvious effect on hematologic functions, except for the increase in fibrinogen and platelets and the slight decrease in circulating RBCs.     

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.     

Presence of factors that activate platelet aggregation in mitral stenotic patients' plasma

BACKGROUND: Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood. METHODS: Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P. RESULTS: There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N. CONCLUSION: Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.