Inhibition of platelet-collagen interaction by propolypeptide of von Willebrand factor

A collagen-binding glycoprotein was isolated from human platelets using affinity chromatography of immobilized collagen. Based upon characterizations of this protein we confirmed that it was identical to the propolypeptide of von Willebrand factor (pp-vWF), which is also called von Willebrand antigen II. The characteristics we have investigated are molecular weight, existence of carbohydrate chains, and the NH2-terminal amino acid sequence. pp-vWF has strong affinity to collagen and inhibits collagen-induced aggregation of human platelets at a concentration as low as 2 micrograms/ml even in the presence of plasma. This inhibitory effect is specific for collagen-induced aggregation since it does not inhibit aggregation of platelets induced by other agonists such as ADP, arachidonic acid, platelet-activating factor, ionophore A23187, and ristocetin. As pp-vWF is quickly released from platelets upon activation by various agonists, it is possible that pp-vWF functions as a repressor for excess platelet aggregation induced by collagen and constitutes a negative feed-back mechanism. Considering the fact that mature vWF supports platelet adhesion to subendothelium, present observations suggest that the propeptide portion and the mature protein could have opposing effects on hemostasis.     

Activation of phospholipases in platelets by polyclonal antibodies against a surface membrane protein.

In a previous paper we demonstrated using immunochemical techniques that propolypeptide of von Willebrand factor was present on the surface of resting platelets. In the present paper we show that polyclonal antibodies against propolypeptide of von Willebrand factor induce activation of phospholipase(s) in platelets and lead to platelet aggregation. The antibody-stimulation of platelets induced the synthesis of thromboxane A2 (TXA2). Furthermore, the aggregation was inhibited by aspirin and an antagonist of TXA2. Aspirin inhibited not only the aggregation but also the activation of arachidonic acid liberation from phospholipids, but the effect of aspirin on arachidonic acid liberation was cancelled by the combined effect of the antibodies and a TXA2 mimetic agonist, which itself did not activate arachidonic acid liberation. The antibody-induced activation of arachidonic acid liberation and the aggregation were blocked by cytochalasin B. All these results obtained with antibodies were quite similar to the results obtained with collagen.     

Exposure of platelet binding sites in von Willebrand factor by adsorption onto polystyrene latex particles

Von Willebrand factor molecules are flexible linear polymers composed of repeating protomeric polypeptide subunits. In the process of primary hemostasis, von Willebrand factor promotes platelet adhesion and platelet plug formation at the site of vascular injury. This biologic activity is apparently related to the multimeric size of von Willebrand factor. We simulated von Willebrand factor binding to the subendothelial surface by adsorbing purified human von Willebrand factor onto polystyrene latex particles of two different diameters, i.e., 0.312 μm and 2.02 μm. The rate and extent of ¹²⁵I-labeled von Willebrand factor binding to polystyrene was similar with both size classes of latex particles. The von Willebrand factor-coated latex beads of 2.02 μm diameter, in contrast to the smaller size, induced rapid agglutination of formalin-fixed human platelets in the absence of any other aggregating agent. Von Willebrand factor was also adsorbed from human plasma onto latex particles coated with anti-von Willebrand factor antibodies. Again, only the large beads, carrying the von Willebrand factor-antibody complex, induced agglutination of fixed platelets. Shear stress promoted the rate of von Willebrand factor adsorption to latex particles. Our results suggest that adsorption to surface exposes binding sites in human von Willebrand factor for platelets.     

Propolypeptide and mature portions of von Willebrand factor of bovine origin recognize different sites on type-I collagen obtained from bovine tendon.

We compared the binding of propolypeptide and mature portions of von Willebrand factor of bovine origin to fibrillar type-I collagen obtained from bovine tendon. The propolypeptide (pp-vWF) and the mature portion (m-vWF) of human origin consist of 741 and 2050 amino acids, respectively, and are rather large proteins. The collagen-binding properties of the two proteins of bovine origin were similar in that both bound more avidly to native collagen than to heat-denatured collagen. Bindings was affected similarly by ionic strength but was not modified either by divalent cations or a synthetic peptide containing Arg-Gly-Asp. However, the binding sites in the fibrillar type-I collagen molecule for pp-vWF and m-vWF seem to be different: the two proteins did not effectively compete with each other for binding to collagen. Furthermore, pepsin treatment of fibrillar type-I collagen resulted in a drastic decrease in the binding of pp-vWF, while only a moderate decrease in the binding of m-vWF was observed after the treatment.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

High pO2-activated inhibitor of protein synthesis in rabbit reticulocytes: its relationship to glutathione disulfide-induced inhibitor and to a approximately 23,000-Mr sulfhydryl protein

Fibronectin is a large dimeric glycoprotein found in plasma, on cell surfaces and as a component of the extracellular matrix which has been implicated in a variety of adhesive processes. The role of fibronectin in platelet function has not been clarified. The present investigation demonstrates that an excess of exogenously added fibronectin inhibits platelet aggregation induced by either thrombin or A23187 at a step subsequent to platelet activation and secretion. Similar concentrations of fibrinogen or von Willebrand factor, both of which also bind to the surface of activated platelets, are not inhibitory. These results are consistent with the concept that fibronectin is one of the important mediators of platelet aggregation.     

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.     

A new role for FcgammaRIIA in the potentiation of human platelet activation induced by weak stimulation

The low affinity receptor for immunoglobulin G, FcgammaRIIA, is expressed in human platelets, mediates heparin-induced thrombocytopenia and participates to platelet activation induced by von Willebrand factor. In this work, we found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in the tyrosine phosphorylation of FcgammaRIIA, through a mechanism involving a Src kinase. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcgammaRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcgammaRIIA. We also found that the anti-FcgammaRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. In addition, thrombin- or U46619-induced tyrosine phosphorylation of several substrates typically involved in FcgammaRIIA-mediated signalling, such as Syk and PLCgamma2, was clearly reduced by incubation with anti-FcgammaRIIA antibody IV.3. Upon stimulation with thrombin, FcgammaRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcgammaRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcgammaRIIA and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcgammaRIIA can be activated in human platelets downstream G-protein-coupled receptors and suggest a novel general mechanism for the reinforcement of platelet activation induced by low concentrations of agonists.     

Collagen-binding domain within bovine propolypeptide of von Willebrand factor

Two reduced/alkylated fragments of bovine propolypeptide of von Willebrand factor (pp-vWF) that inhibit pp-vWF binding to collagen were isolated. One is a tryptic fragment of molecular mass of about 30 kDa and inhibits the binding at a molar concentration about 20 times higher than the intact pp-vWF. Amino acid sequence of this fragment was determined almost completely, and it was revealed that this fragment corresponded to the carboxyl-terminal region of pp-vWF molecule beginning with Phe557. The other active fragment was obtained by lysyl endopeptidase digestion. This migrated as a 21.5/21-kDa doublet in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but deglycosylation of this doublet resulted in production of single species of 19 kDa. The difference between the doublet constituents, therefore, was of carbohydrate composition. The extent of inhibition of collagen-binding by this 21.5/21-kDa fragment was comparable to that of the 30-kDa fragment, and furthermore, location of this fragment in the molecule was established to be between Phe570 and Lys682. These were the only fragments among those obtained by proteolytic digestions that had significant competitive effect on the binding of intact pp-vWF to collagen. These results strongly suggest that at least one collagen-binding site should be present in the carboxyl-terminal region of bovine pp-vWF extending from residue 570 to 682.     

A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor.

Propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen, and we have reported that a binding domain exists in a 21.5/21-kDa fragment originated from the C-terminal portion [Takagi, J., Fujisawa, T., Sekiya, F., & Saito, Y. (1991) J. Biol. Chem. 266, 5575-5579]. The collagen-binding property of the 21.5/21-kDa fragment was compared with that of the intact pp-vWF. Although pp-v WF preferentially binds to native type I collagen fibrils, the isolated fragment no longer has this specificity and binds well to collagen of other types in the native and heat-denatured states. In order to determine the critical site that mediates this collagen/gelatin binding, several peptides were synthesized based on the primary structure of the 21.5/21-kDa fragment. Among these, a 25-residue peptide strongly inhibited the binding of the 125I-labeled 21.5/21-kDa fragment to collagen. Using this inhibitory effect as an index, the binding site was defined to the sequence as follows: WREPSFCALS. Furthermore, a decapeptide of this sequence bound to collagen and gelatin, indicating that this sequence is responsible for the binding of the 21.5/21-kDa fragment to collagen/gelatin.     

Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[alpha(32)P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[alpha(32)P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca(2+) flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.     

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.     

Ability of Different Glycoprotein IIb-IIIa Ligands to Support Platelet Aggregation Induced by Activating Antibody CRC54

The ability of different ligands of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3-integrin) to support platelet aggregation stimulated by activating anti-GP IIb-IIIa monoclonal antibody (monoAB) CRC54 has been investigated. Antibody CRC54 stimulated aggregation of washed platelets not only in the presence of fibrinogen, the main GP IIb-IIIa ligand, but also in the presence of von Willebrand factor (vWF). Unlike these ligands, fibronectin failed to support CRC54-induced aggregation. Fibrinogen and vWF dependent platelet aggregation was completely suppressed by GP IIb-IIIa antagonists--preparations Monafram (F(ab')2 fragments of monoAB that blocked GP IIb-IIIa receptor activity) and aggrastat (RGD-like peptidomimetic). However, aggregation stimulated in the presence of vWF was also completely inhibited by monoAB AK2 directed against GP Ib and capable of blocking its binding with vWF. CRC54-induced aggregation of platelets from patient with GP Ib deficiency in the presence of vWF was significantly lower than aggregation of platelets from normal donors and was not inhibited by anti-GP Ib antibody but still blocked by GP IIb-IIIa antagonist Monafram. Monafram also suppressed CRC54-stimulated platelet adhesion to plastic-adsorbed fibrinogen, vWF, and fibronectin. Unlike CRC54-induced platelet aggregation supported by fluid phase vWF, CRC54-induced adhesion to adsorbed vWF was not affected by anti-GP Ib antibody. Aggregation induced by CRC54 in the presence of fibrinogen and vWF was only partially suppressed by prostaglandin E1, an inhibitor of platelet activation, and was associated with serotonin release from platelet granules only when Ca2+ concentration was decreased from 1 mM (physiological level) to 0.1 mM. The data indicate that vWF supports CRC54-induced platelet aggregation via interaction with two receptors--GP IIb-IIIa and GP Ib. Aggregation induced by CRC54 in the presence of vWF or fibrinogen is only partially dependent on platelet activation and is accompanied with granule secretion only at low Ca2+ concentrations.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

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.     

Biosynthesis of major platelet proteins in human blood platelets

We studied de novo protein biosynthesis in platelets of normal adult donors and in newly formed platelets isolated from splenectomized patients with idiopathic thrombocytopenic purpura (ITP). After metabolic labelling of platelet proteins, performed with different radiolabelled amino acids or carbohydrates, a tenfold increase in incorporation of radioactivity into trichloroacetic-acid-precipitable material was obtained with ITP platelets compared to control platelets. Electron microscopic studies of ITP platelets revealed the presence of rough endoplasmic reticulum and polyribosomes, providing morphological evidence for protein synthesis. SDS-PAGE of radiolabelled ITP platelet proteins followed by autoradiography showed that [35S]methionine and [3H]leucine were incorporated into almost all Coomassie-blue-stained proteins whereas [3H]carbohydrates only labelled a few bands. Using crossed-immunoelectrophoresis we identified some of the labelled platelet compounds and demonstrated that major membrane glycoproteins (GPIb, IIb, IIIa) and alpha-granule proteins, such as fibrinogen, thrombospondin, albumin and von Willebrand factor, were synthesized in newly formed circulating platelets.     

A collagen-binding glycoprotein from bovine platelets is identical to propolypeptide of von Willebrand factor

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.     

The inhibition of platelet-activating factor-induced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism

In an earlier study (Miwa, M., Hill, C., Kumar, R., Sugatani, J., Olson, M. S., and Hanahan, D. J. (1987) J. Biol. Chem. 262, 527-530) it was shown that an inhibitor of platelet-activating factor (PAF), a powerful endogenous mediator of platelet aggregation, was present in freeze-clamped perfused livers. Subsequently, we determined that this substance was a mixture of unsaturated free fatty acids (FFA). Among these FFA, oleic acid between 10 and 100 microM was found to be a potent inhibitor of PAF-induced platelet aggregation and serotonin secretion. Consequently, in order to understand the molecular mechanism of oleic acid action, we investigated the effects of this FFA on several biochemical events associated with platelet aggregation induced by PAF. The effect of oleic acid and/or PAF on the level of [32P]phosphatidylinositol 4-phosphate (PIP) and [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) was examined by using platelets labeled with [32P]phosphate. Oleic acid induced a dose-dependent decrease in the levels of [32P]PIP and [32P]PIP2; a maximal decrease in [32P]PIP and [32P]PIP2 of approximately 50 and 25%, respectively, was observed within seconds after the addition of 20 microM oleic acid and persisted for at least 15 min. Oleic acid did not induce the formation of [3H]inositol phosphates in platelets prelabeled with [3H]inositol, suggesting that the decrease in [32P]PIP and [32P]PIP2 was not due to a stimulation of phospholipase C. In contrast to oleic acid, PAF induced a dose-dependent increase in the [32P]PIP level, reaching a maximum of approximately 200% 3 min after the addition of 1 nM PAF to the platelets. This increase in [32P]PIP was accompanied by platelet aggregation and secretion, and a close correlation was established between the [32P]PIP level and the degree of aggregation. Oleic acid and PAF, when added together to the platelets, interacted by affecting the level of [32P]PIP and [32P]PIP2 in an opposite way since the decrease in the level of [32P]PIP and [32P] PIP2 induced by oleic acid was partially reversed by an excess of PAF. The decrease in the levels of [32P] PIP and [32P]PIP2 caused by oleic acid was associated with an inhibition of platelet aggregation induced by PAF. Interestingly, oleic acid did not block [3H]PAF binding to platelets but inhibited the PAF-induced phosphorylation of platelet proteins of 20 kDa and 40 kDa. These results suggest that inhibition of the PAF response by oleic acid may be at one of the steps in the signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.     

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm² and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.