Platelet-collagen Interactions Research Articles

Heat Shock Protein 47: A New Platelet Collagen Receptor

AbstractAbstract 158We recently reported that Heat shock protein 47 (Hsp47), a well established collagen binding protein, was present on the surface of human platelets. In the current study we demonstrate that anti-Hsp47 antibodies and a small molecule inhibitor of Hsp47 selectively prevent aggregation of platelets induced with collagen fibrils. Inhibition of Hsp47 was also found to reduce the size of thrombi produced upon perfusion of blood under arterial flow conditions over collagen in vitro. Inclusion of Integrilin, to prevent platelet-platelet interactions via fibrinogen, indicated that the reduced size of thrombi was due to fewer direct platelet-collagen interactions. Collagen-induced tyrosine phosphorylation of Syk, an early marker of signalling from the collagen receptor GPVI was unaffected by the Hsp47 inhibitor, suggesting that any contribution to platelet function by Hsp47 may be independent of GPVI-proximal signalling. Surface plasmon resonance analysis of recombinant his-tagged human Hsp47 revealed its ability to interact directly with collagen fibrils, an interaction that was disrupted by the small molecule inhibitor of Hsp47.Mouse platelets were also found to contain Hsp47, and aggregation induced with collagen was reduced by inhibitors of Hsp47. Infusion of Hsp47 inhibitor into mice increased tail bleeding times and volumes of blood lost, even in the absence of anticoagulant. This effect was likely due to inhibition of platelet function, since the thrombi formed in cremaster muscle arterioles using an intravital laser-injury model of thrombosis were smaller following administration of the Hsp47 inhibitor.Together these data indicate Hsp47 to function on the platelet surface as a collagen receptor that potentially represents a new target for antithrombotic therapy. Disclosures:No relevant conflicts of interest to declare.

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.

Aegyptin displays high‐affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo

Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light-scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high-affinity binding site for aegyptin, with a K(D) of approximately 5 nM. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat-denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO)(10) and GFOGER with low affinity (microM range), which respectively represent glycoprotein VI and integrin alpha2beta1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo.

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

AbstractCarcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)–γ-chain. ceacam1−/− platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1−/− mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1−/− mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1−/− mice showed a reversal in the more stable thrombus growth phenotype. ceacam1−/− mice were more susceptible to type I collagen–induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.

Microfluidic Flow Studies of Murine Thrombus Formation and Stability on Collagen.

Glycoprotein (GP) VI and integrin α2β1 are platelet receptors for collagen which, along with GPIb and von Willebrand factor, mediate platelet adhesion to sites of vascular injury. In pathologic states, these receptors may cause inappropriate platelet activation resulting in thrombosis. The primary objective of this study was to examine in real time, the relative contribution of α2β1 and GPVI, in platelet aggregation and thrombus stability on collagen under flow. A novel microfluidic flow chamber device was used which allowed the effects of a single agonist to be studied under varying shear rates using just 50μL of whole blood. In addition, real-time imaging of the flow experiment allowed the dynamics of thrombus formation to be observed in response to a platelet agonist, thus simulating vascular injury. In the microfluidic flow experiments, we used whole blood anti-coagulated with the thrombin inhibitor PPACK to study the accumulation of mouse platelets in response to fibrillar and acid-soluble collagen patterned in a 100μm square on a glass slide. By tagging platelets with fluorescent anti-CD41 antibodies, it was possible to quantify the degree to which platelets adhere and aggregate by measuring the fluorescence intensity, corresponding to thrombus size, with time. After allowing a thrombus to form for 300 seconds at a shear of 400s−1, the shear in the chamber was increased to 8000s−1 to assess the stability of the platelet aggregate under high shear. This served as a method for evaluating the strength of platelet-platelet and platelet-collagen interactions. The three types of knockout mice used in the evaluation of the roles of these receptors in platelet-collagen interaction were α2−/− GPVI−/− and α2−/−/FcRγ−/−. The α2 knockout eliminates the α2β1 receptor while the FcRγ knockout deletes FcRγ and prevents its partner GPVI from being expressed on the cell surface. The data from these knockout mice were compared to measurements from wild type mice and major differences in thrombus formation and stability were identified. Platelets from α2−/−/FcRγ−/− double knockout mice showed a markedly diminished ability to adhere to both acid-soluble and fibrillar types of collagen as compared to wild type mice. The small aggregates that formed washed away rapidly in response to an increase in shear. Platelets from α2−/− mice exhibited significantly less thrombus formation than wild type, but adhered and aggregated to a greater extent than platelets from α2−/−/FcRγ−/− double knockout mice. There was no significant difference in aggregation of α2−/− platelets on both types of collagen. Additionally, α2−/− thrombi were slightly more stable than α2−/−/FcRγ−/− thrombi, as evidenced by the small aggregate that remained following an increase in shear. GPVI−/− platelets also produced smaller aggregates than wild type, but they were larger than those produced by either α2−/−/FcRγ−/− or α2−/− platelets. Although GPVI−/− thrombi were less stable than wild type, significant thrombus remained following an increase in shear suggesting that α2β1, not GPVI, may be the most important collagen receptor for maintaining thrombus strength. While these findings differ from those commonly supported by the literature, the small diameter of the microfluidic flow chamber may better approximate the flow conditions in arterioles than traditional flow chambers. In conclusion, these data show a significant decrease in thrombus formation and stability for α2−/− and α2−/−/FcRγ−/− platelets and a moderate decrease in aggregation of GPVI−/− platelets with minimal decrease in thrombus stability as compared to wild type. These results suggest that α2β1 plays a critical role in adhesion and aggregation of platelets to Type I collagen under flow.

Transdermal 17-β estradiol replacement therapy reduces megakaryocyte GPVI expression

The platelet-collagen interaction is a critical early event in arterial thrombus formation, and platelet GPVI is the major activating receptor for collagen. We have previously used a mouse model to demonstrate that the estrogen effects on platelets depend upon the agonist, estrogen formulation and route of administration. In the current study we used a model of transdermal estradiol (E2) administration to ovariectomized mice to address the potential inhibitory effects of E2 on platelet GPVI. Platelet GPVI expression was reduced after transdermal E2 replacement therapy (p ≤ 0.001) but no evidence of GPVI shedding was found when platelets were directly incubated with E2. In addition, significantly reduced GPVI-mediated fibrinogen binding and aggregation were observed in platelets from mice subjected to 9 days or longer of in vivo E2 treatment, but not in platelets from mice treated for 3 days or shorter, suggesting an indirect pathway. Studies with mouse bone marrow revealed that E2 replacement in ovariectomized mice reduces megakaryocyte GPVI expression. This data suggest that transdermal E2 is able to affect centrally on megakaryocyte GPVI to regulate platelet GPVI and function.

Abstract 1007: Platelet Glycoprotein VI (GPVI) Deletion Reduces Myocardial Infarction in Mice

Fibrillar collagen, exposed by damage of vascular endothelium, is the most thrombogenic component of the subendothelium, which, in addition to supporting platelet adhesion, is also a potent platelet activator. Platelet glycoprotein VI (GPVI) plays a major role in collagen-induced platelet activation, leading to thrombus formation. Inhibition of GPVI function may be a novel anti-platelet therapy with minimal bleeding risk. Recent evidence in FcRγ-chain-knockout (KO) mice, which also lack GPVI, shows attenuated myocardial infarction after ischemia and reperfusion. Unlike GPVI, which is exclusively expressed in platelets and megakaryocytes, FcRγ-chain also exists in white blood cells and immune cells. Thus, it is unclear whether GPVI deletion is cardioprotective. In this study, we compared myocardial infarction in GPVI-KO and age-matched wild-type (WT) mice. In parallel FcRγ-chain-KO mice were compared with age-matched WT mice. A major branch of left coronary artery was ligated for 30 min followed by reperfusion for 24 hrs. Myocardial infarction was measured by tetrozolium staining. Hemodynamics were not statistically different among the groups. Risk zone sizes were also similar between WT and GPVI-KO mice (0.020 ± 0.004 cm 3 and 0.022 ± 0.005 cm 3 , respectively) (Mean ± SD). However, percentage infarction of the risk zone was significantly smaller in GPVI-KO mice (averaged 22 ± 8 % vs. 45 ± 18 % in WT). Comparable protection was observed in FcRγ-chain-KO mice (12 ± 6 % vs. 46 ± 13 % in WT). Significant exposure of vascular collagen in the ischemia/reperfusion region of myocardium was revealed by FITC-labeled recombinant GPVI protein, injected prior to ischemia, indicating endothelium injury. Furthermore, there was significantly less expression of P-selectin, a marker of activated platelets, in the myocardium from GPVI-KO and FcRγ-KO mice when compared with WT mice. It is concluded that deletion of GPVI reduces myocardial infarction induced by ischemia and reperfusion, likely due to the attenuation of the platelet-collagen interaction. With its known anti-thrombotic effect and minimal risk of bleeding in animals, anti-GPVI therapy may offer a promising novel treatment for cardiovascular diseases.

Aegyptin, a Novel Mosquito Salivary Gland Protein, Specifically Binds to Collagen and Prevents Its Interaction with Platelet Glycoprotein VI, Integrin α2β1, 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.

Peptides Derived from Platelet Non-integrin Collagen-receptors or Types I and III Collagen Inhibit Collagen-Platelet Interaction

Platelet-collagen interaction plays an important role in hemostasis and pathological thrombosis. Upon an injury to the subendothelium of a blood vessel wall, platelets adhere to the denuded substrate, aggregate, and release biological substances. Many investigators have explored the use of blocking agents to interrupt the final step of binding fibrinogen on glycoprotein (GP) IIb/IIIa of activated platelets. A potent peptide is Arg-Gly-Asp-Ser (RGDS) and its derivatives in various forms. Results from many clinical trials show that the efficacy of these antagonists does not lie in blocking the adhesion of platelets to the distal site(s) of the injury as expected. Because type I and type III collagens are predominant components of blood vessel walls, other laboratories and ours have defined various active peptides from either collagen molecules or platelets as useful for blocking collagen-platelet interaction. An active hybrid peptide derived from both platelet types I and type III collagen receptors that abolishes type I and type III collagen-induced platelet aggregation has been obtained. The hybrid peptide inhibits the binding of type I and type III collagens to washed human platelets, platelet aggregation, and the adhesion of washed platelets to rabbit aortic segments. However, the usefulness of the defined hybrid peptide in preventing thrombi formation in vivo requires further investigation.

The leech product saratin is a potent inhibitor of platelet integrin α2β1 and von Willebrand factor binding to collagen

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and alpha2beta1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet-collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking alpha2beta1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated alpha2beta1-dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet-rich plasma adhesion to fibrillar collagen, a process dependent upon both alpha2beta1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the alpha2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and alpha2beta1. These results demonstrate that saratin interferes with integrin alpha2beta1 binding to collagen in addition to inhibiting von Willebrand factor-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.

The Leech Product Saratin Is a Potent Inhibitor of Both VWF and Integrin α2β1 Binding to Collagen.

Background: Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor (VWF), which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors GPVI and α2β1 to collagen is critical for platelet activation and stable adhesion. Hematophages, such as leeches, have evolved a number of inhibitors directed toward platelet-colagen interactions so as to prevent haemostasis in the host during haematophagy.Objective: To elucidate the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen.Results: In the presence of inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), both saratin and 6D1, a blocking α2β1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated α2β1-dependent platelet adhesion to soluble collagen in the presence of a Src kinase inhibitor. Moreover, saratin prevented platelet-rich-plasma adhesion to fibrillar collagen, a process dependent upon both α2β1 and VWF binding to collagen. Furthermore, saratin specifically inhibited the binding of the α2I-domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of an anti-GPIb and anti-α2β1 mAb.Conclusions: The present study demonstrates that saratin interferes with integrin α2β1 binding to collagen in addition to inhibiting VWF-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.

The role of platelets in the initial stages of atherosclerosis.

The role of platelets in the initial stages of atherosclerosis.

Function of GP Ia/IIa in the platelet-collagen interactions

目的 探讨GP Ⅰa/Ⅱa在血小板-胶原黏附过程和胶原诱导血小板颗粒释放反应中的功能.方法采用流式细胞术,在有或无抗GP Ⅰa/Ⅱa抗体阻断条件下,(1)用anti-CD42b-PE标识血小板并检测血小板与FITC荧光标记胶原黏附过程中FITC荧光强度的变化;(2)用anti-CD61-PerCP标识血小板并用PE标记的抗CD62P抗体检测胶原诱导血小板颗粒释放反应中CD62P的变化.结果静息加6F1组比静息组下降9.0%,活化加6F1组比活化组降低33.6%.抗GP Ⅰa/Ⅱa抗体能抑制血小板与胶原的黏附,对活化态血小板的抑制作用更明显(P＜0.01);抗GPⅠa/Ⅱa抗体不能抑制胶原诱导的血小板释放反应(P＞0.05).结论GP Ⅰa/Ⅱa在血小板与胶原黏附过程中有重要作用,阻断GPⅠa/Ⅱa能降低血小板对胶原的结合,但阻断GP Ⅰa/Ⅱa难以抑制由胶原诱导的血小板颗粒内容物的释放。

Type I and type III collagen–platelet interaction: Inhibition by type specific receptor peptides

We have previously cloned and characterized a platelet receptor for type III collagen (47 kDa) from a human bone marrow cDNA phage library and defined two active peptides. We also cloned and characterized a platelet receptor for type I collagen (65 kDa) and defined an active peptide. Our objective was to study whether there is type specificity of these active peptides. We have engineered a mutant receptor clone by replacing one of the two active peptides of the platelet receptor for type III collagen with the active peptide of the platelet receptor for type I collagen. The replacement of an active peptide at the amino terminal end (rMIII) of the platelet receptor for type III collagen with the type I collagen active peptide was done without altering the hydrophilicity of the protein. This purified recombinant protein reacts with polyclonal anti-47-kDa and anti-65-kDa active peptide antibodies. The purified recombinant protein inhibits both types I and III collagen-induced platelet aggregation. This rMIII also inhibits the adhesion of washed platelets to rabbit aortic segments (natural matrix) in a dose-dependent manner. The chemically synthesized hybrid peptide of each active peptide of platelet type I and type III collagen receptors inhibits types I and III collagen-induced platelet aggregation in a dose-dependent manner. These results suggest that there is a type specific reactive site on platelets for type I and type III collagens.

Collagen-induced platelet activation

Platelet collagen receptors, such as Gp VI, are attractive targets for antithrombotic therapy. In this paper, I discuss the current knowledge regarding collagen–platelet interactions, including the role of platelet receptors, the recognition of collagen by platelets, the effect of the interaction on platelet activation and thrombosis and the effect of collagen structure on the platelet interaction, and highlight the areas in which additional information is required to pursue the goal of antithrombotic therapy, using the collagen–platelet interaction as the site of intervention. Understanding the detail of the receptor recognition motifs within collagen not only may reveal new antithrombotic targets within collagen, but will almost certainly lead to the development of defined reagents that can be used in vitro and ex vivo to explore thrombus formation further.

GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism

Platelet glycoprotein VI (GPVI) is now considered to be a major player in platelet–collagen adhesive interactions leading to thrombus formation. GPVI blockade, or its depletion, has been shown in mice to result in complete protection against arterial thrombosis, without significant prolongation of bleeding time. GPVI may therefore represent a useful antithrombotic target. In order to reaffirm the role of GPVI in platelet–collagen interactions, we developed GPVI null mice by targeted disruption methodology. GPVI null mice platelets failed to respond to a high dose of fibrillar collagen, or convulxin, a GPVI agonist, but showed a normal response to other agonists such as ADP, PMA and arachidonic acid. We report, for the first time, that a proportion of GPVI null mice is protected against lethal thromboembolism, induced by the infusion of a mixture of collagen and epinephrine. Greater than 55% of GPVI null mice survived the challenge, whereas the maximal survival from the other genotypes was 17% ( n = 18 per genotype). Washed platelets obtained from GPVI null mice showed > 90% reduction in adhesion to fibrillar collagen under static conditions. Platelet adhesion to collagen under dynamic conditions using a high shear rate (2600 s − 1 ) was dramatically reduced using blood from GPVI null mice, while platelets from wild-type and heterozygous animals showed a similar amount of adhesion. Animals from each genotype had essentially similar tail bleeding time, suggesting that a complete deficiency of GPVI, at least in mice, does not result in an enhanced bleeding tendency. These observations clearly establish that blockade of GPVI may attenuate platelet–collagen interactions without adversely affecting the bleeding time.

Evidence for a Role of ADAM17 (TACE) in the Regulation of Platelet Glycoprotein V

Glycoprotein V (GPV) is a subunit of the GPIb-IX-V receptor for von Willebrand factor and thrombin and has been shown to modulate platelet responses to the two strongest physiological agonists, thrombin and collagen. Thrombin directly cleaves GPV from the platelet surface, yielding a 69-kDa fragment GPV f1 of unknown function. We show here that a approximately 82-kDa fragment of GPV is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide. This shedding was inhibited by the broad range metalloproteinase inhibitor GM6001, the two potent ADAM17 inhibitors GW280264X and TAPI-2, and was absent in mice lacking functional ADAM17 (ADAM17 lacking Zn-binding domain; ADAM17(DeltaZn/DeltaZn)). Furthermore, we show that recombinant ADAM17 ectodomain efficiently releases GPV from the platelet surface. GPV is known to be associated with the intracellular regulatory protein calmodulin, which has previously been shown to be involved in ADAM17-mediated shedding of l-selectin from the surface of leukocytes. As in these reports, inhibition of calmodulin led to rapid GPV shedding from the platelet surface, a process that was again blocked by GM6001 or ADAM17 inhibitors and that was absent in ADAM17(DeltaZn/DeltaZn) mice. Inhibition of outside-in signaling through GPIIb/IIIa did not significantly affect GPV shedding, excluding an essential role of this pathway for the regulation of ADAM17 activity. These results demonstrate that GPV is cleaved upon agonist-induced platelet activation and show that ADAM17 is the major enzyme mediating this process.

Current methods of assessing platelet function: relevance to transfusion medicine

Current methods of assessing platelet function: relevance to transfusion medicine

The early discoveries of collagen–platelet interaction and studies on its role in hemostatic plug formation

In the recently published book on Platelets in Thrombotic and Non-Thrombotic Disorders it is stated in Chapter 11 [1] that ‘collagen–platelet interaction is fundamental to normal hemostasis and pathological thrombotic incidents’. Nice, then, to recall that more than 40 years ago we contributed a small stone to the foundation of a large construction which has proved to be very complicated.

A defined peptide that inhibits the formation of the glycoprotein IIb and IIIa complex

Collagen–platelet interaction plays an important role in hemostasis and pathological thrombosis. The proposed mechanism of the interaction was the activation of platelets→releasing of contents from granules→aggregation. The common end point is the platelets and fibrin aggregates. Platelet glycoprotein (GP) IIb/IIIa (the αIIbβ3 integrin) complexes serve as a receptor for the binding of fibrinogen to form firmed aggregates. Blockading of GP IIb/IIIa has been proposed to prevent platelet aggregation independent of the substance(s) responsible for activating the platelets. The development of various forms of GP IIb/IIIa inhibitor has resulted in the inhibition of platelet aggregation, although studies of αIIbβ3 receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function as well as having nonplatelet effects. This study investigated platelet inhibition provided by blocking the GP IIb/IIIa complex formation by using a peptide derived from the GP IIIa molecule. The peptide inhibits both types I and III collagen-induced platelet aggregation in a dose-dependent manner. The defined peptide interferes with the formation of the GP IIb/IIIa complex by inhibiting the binding of FITC–PAC-1 onto ADP-, type I collagen-, and type III collagen-activated platelets. However, P-selectin secretion is not affected by the peptide. In addition, the peptide is not interfering with the binding of FITC–PAC-1 to platelets that were preincubated with indomethacin. Results from this study may suggest that the defined peptide is an effective agent to block the interaction of types I and III collagen with platelets.