Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen.

The integrin alpha(2)-subunit was ablated in mice by targeted deletion of the ITGA2 gene. alpha(2)-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin alpha(2)beta(1) has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, alpha(2)-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of alpha(2)-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, alpha(2)-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of alpha(2)-deficient but not wild type platelets is abrogated. These results demonstrate that integrin alpha(2)beta(1) significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of alpha(2)-deficient platelets to soluble collagen. Thus, alpha(2)beta(1) plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that alpha(2)beta(1)-deficient animals suffer no bleeding anomalies.     

Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor

Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this study we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I-V (K(d) approximately 1 nm) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings suggest that aegyptin recognizes specific binding sites for glycoprotein VI, integrin alpha2beta1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.     

Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP VI

alpha2beta1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha2beta1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha2beta1 integrin antibody. The combination of anti-alpha2beta1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha2beta1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha2beta1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha2beta1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.     

Inhibition of collagen-induced platelet reactivity by DGEA peptide

Direct interactions between collagen, the most thrombogenic component of the extracellular matrix, and platelet surface membrane receptors mediate platelet adhesion and induce platelet activation and aggregation. In this process two glycoproteins are crucial: integrin alpha2beta1, an adhesive receptor, and GPVI, which is especially responsible for signal transduction. Specific antagonists of the collagen receptors are useful tools for investigating the complexity of platelet-collagen interactions. In this work we assessed the usefulness of DGEA peptide (Asp-Gly-Glu-Ala), the shortest collagen type I-derived motif recognised by the collagen-binding integrin alpha2beta1, as a potential antagonist of collagen receptors. We examined platelet function using several methods including platelet adhesion under static conditions, platelet function analyser PFA-100TM, whole blood electric impedance aggregometry (WBEA) and flow cytometry. We found that DGEA significantly inhibited adhesion, aggregation and release reaction of collagen activated blood platelets. The inhibitory effect of DGEA on static platelet adhesion reached sub-maximal values at millimolar inhibitor concentrations, whereas the specific blocker of alpha2beta1 - monoclonal antibodies Gi9, when used at saturating concentrations, had only a moderate inhibitory effect on platelet adhesion. Considering that 25-30% of total collagen binding to alpha2beta1 is specific, we conclude that DGEA is a strong antagonist interfering with a variety of collagen-platelet interactions, and it can be recognised not only by the primary platelet adhesion receptor alpha2beta1 but also by other collagen receptors.     

The domain of platelet glycoprotein I as recognized by various antibodies, both monoclonal and polyspecific

Platelet membrane glycoprotein Ib plays a major role in the binding of factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium. We have used polyspecific and monoclonal antibodies to glycoprotein Ib and have demonstrated that both antibodies were directed to glycoprotein Is, a soluble fragment of glycoprotein Ib. By showing an inhibition of the binding of factor VIII/von Willebrand factor to control platelets in presence of the antibodies, it can be concluded that glycoprotein Is is involved in these binding sites.     

RhoA sustains integrin alpha IIbbeta 3 adhesion contacts under high shear

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, alpha(IIb)beta(3). Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin alpha(IIb)beta(3), by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin alpha(IIb)beta(3)-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase had no effect on integrin alpha(IIb)beta(3) activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin alpha(IIb)beta(3) adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin alpha(IIb)beta(3) adhesion contacts under conditions of high shear stress.     

Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2

Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet-collagen interaction, adhesion is mediated via the inside-out regulation of integrin alpha2beta1 and activation through the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. The present study extends this model by demonstrating that engagement of alpha2beta1 by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase-based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI-FcR gamma-chain complex. The same events do not occur in platelet suspensions. alpha2beta1 activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI-FcR gamma-chain cascade, including Src, Syk, SLP-76, and PLCgamma2 as well as plasma membrane calcium ATPase and focal adhesion kinase. alpha2beta1-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLCgamma2-deficient platelets. Spreading is abolished by chelation of intracellular Ca2+. Demonstration that adhesion of platelets to collagen via alpha2beta1 generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of beta1-deficient thrombi and why loss of the GPVI-FcR gamma-chain complex has a relatively minor effect on bleeding.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Redundant mechanism of platelet adhesion to laminin and collagen under flow: involvement of von Willebrand factor and glycoprotein Ib-IX-V

Although the role of collagen in thrombosis has been extensively investigated, the contribution of other extracellular matrices is still unclear. We have recently reported that laminin stimulates platelet spreading through integrin alpha(6)beta(1)-dependent activation of the collagen receptor glycoprotein (GP) VI under static condition. Under physiological high and low shear conditions, platelets adhered to laminin, and this was strongly inhibited by an antibody that blocks association between GPIb-IX-V and von Willebrand factor (VWF). Moreover, platelets of type III von Willebrand disease or Bernard-Soulier syndrome adhered to laminin at a low shear condition but not at a high shear condition. The specific binding of laminin to VWF was confirmed by surface plasmin resonance spectroscopy (BIAcore). These findings suggest that laminin supports platelet adhesion depending on the interaction of VWF and GPIb-IX-V under pathophysiological high shear flow. This mechanism is similar to that of collagen. We propose that integrins, GPVI, GPIb-IX-V, and VWF represent a general paradigm for the interaction between platelets and subendothelial matrices.     

Differential Involvement of ERK2 and p38 in platelet adhesion to collagen

We investigated the role of two MAP kinases, ERK2 and p38, in platelet adhesion and spreading over collagen matrix in static and blood flow conditions. P38 was involved in collagen-induced platelet adhesion and spreading in static adhesion conditions, whereas ERK2 was not. In blood flow conditions, with shear rates of 300 or 1500 s(-1), ERK2 and p38 displayed differential involvement in platelet adhesion, depending on the presence or absence of the von Willebrand factor (vWF). Low collagen coverage densities (0.04 microg/cm2) did not support vWF binding. During perfusions over this surface, platelet adhesion was not affected by the inhibition of ERK2 phosphorylation by PD 98059. However, abolishing p38 activation by SB 203580 treatment reduced platelet adhesion by 67 +/- 9% at 300 s(-1) and 56 +/- 2% at 1500 s(-1). In these conditions, the p38 activity required for platelet adhesion depends on the alpha2beta1 collagen receptor. At higher collagen coverage densities (0.8 microg/cm2) supporting vWF binding, the inhibition of ERK2 activity by PD 98059 decreased adhesion by 47 +/- 6% at 300 s(-1) and 72 +/- 3% at 1500 s(-1), whereas p38 inhibition had only a small effect. The ERK2 activity required for platelet adhesion was dependent on the interaction of vWF with GPIb. In conclusion, ERK2 and p38 have complementary effects in the control of platelet adhesion to collagen in a shear stress-dependent manner.     

Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb

To identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr approximately 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein IIb alpha, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins IIb and III form a complex. Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited greater than 80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein IIb participates in some aspect of platelet-collagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of approximately 25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein IIb is not the only molecule involved in this process.     

Efficient Inhibition of Collagen-Induced Platelet Activation and Adhesion by LAIR-2, a Soluble Ig-Like Receptor Family Member

LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s⁻¹; IC₅₀ = 18 µg/ml) and high shear rate (1500 s⁻¹; IC₅₀ = 30 µg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and α2β1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis.     

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.     

Convulxin binds to native,human glycoprotein Ib alpha

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.     

A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

Identification of the disulphide bonds in human platelet glycocalicin

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.     

Platelet membrane glycoproteins and their function: an overview

The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.     

Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.