We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5 end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to Fc␣R and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fc␥ and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca 2؉ in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.The adhesion and activation of resting, circulating platelets at a site of vascular injury is the first step in a process leading to the formation of a thrombus, which is converted into a hemostatic plug. Collagen is one of the major components of the vessel wall responsible for platelet activation. Many types of collagen exist, and seven of these are found in the subendothelial layers. Several different receptors for collagen have been identified on platelets including CD36 (1) and a p65 collagen type I specific receptor (2), but the major ones are now considered to be the integrin ␣ 2 ␤ 1 and the nonintegrin glycoprotein (GP) 1 IV. Although ␣ 2 ␤ 1 is well characterized and both subunits were cloned and sequenced several years ago (3, 4), the structure of GPVI has remained elusive; however, several features have been identified. It was determined about 20 years ago that GPVI is a major platelet glycoprotein with a molecular mass in the 60 -65-kDa range and an acid pI (5). Its role as a putative collagen receptor was established following the identification of a patient in Japan with a mild bleeding disorder whose platelets had a specific defect in response to collagen and lacked this receptor (6). This patient had also developed autoantibodies to the deficient receptor, and these were used to characterize the molecule f...

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxininduced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of timedependent tyrosine phosphorylation of platelet proteins, and binding studies with 125 I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor ␥ chain, phospholipase C␥2, p72 SYK , c-Cbl, and p36 -38. However, unlike collagen, pp125 FAK and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72 SYK , showed differential effects on collagen and convulxinstimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the ␣ 2 ␤ 1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor ␥ chain and, by a mechanism previously described in T-and B-cells, activate p72 SYK that is critical for downstream activation of platelets.A large number of C-type lectins from snake venoms have been described over the last few years with effects on hemostasis. While most of these inhibit the function of the coagulation factors and platelet components that they bind to, a few activate platelets by direct or indirect effects. So far all of these have been shown to affect the von Willebrand factor (vWf) 1 -platelet GPIb-V-IX axis. They include botrocetin (1) and bitiscetin (2) that bind to and change the conformation of vWf so that it can bind to GPIb and thus activate platelets and alboaggregin B (3) that activates platelets directly by binding to, and presumably clustering, GPIb. A further snake peptide from the venom of some Crotalus species has subunits with a molecular mass similar to the C-type lectins, is a strong activator of platelet phospholipase C, and has been termed convulxin (4 -8). We have isolated a similar, possibly identical, molecule from Crotalus durissus terrificus venom and show that it belongs to the heterodimeric, C-type lectin family. It activates platelets not via GPIb but through the p62/GPVI component of the platelet collagen receptor, probably by a clustering effect, and induces signals similar to a ...

To cite this article: Polgar J, Matuskova J, Wagner DD. The P-selectin, tissue factor, coagulation triad. J Thromb Haemost 2005; 3: 1590-6.Summary. The primary importance of tissue factor (TF) in blood coagulation and thrombus propagation has been recognized for many years. Nevertheless, our view about the origin of TF activity, necessary for normal hemostasis and found in pathologic conditions, needs to be revised in the light of recent observations. Pioneering work by Yale Nemerson's group showed that circulating TF on microparticles (MPs), could promote thrombus growth. The origin and characteristics of this Ôblood-borneÕ TF are targets of intense research as well as intense debate. Surprising observations now implicate the adhesion receptor P-selectin (P-sel), known for its role in inflammation, in these MPsÕ generation. P-sel, translocated from granules to the cell surfaces of activated platelets and endothelial cells, was recently found to play multiple roles in hemostasis. Expressed on endothelium, it can mediate platelet rolling. Signaling by P-sel through its receptor on leukocytes, P-selectin glycoprotein ligand 1 (PSGL-1), induces the generation of TF-positive, highly procoagulant MPs. In addition, P-sel on activated platelets helps to recruit these MPs specifically to thrombi. In this review, we discuss the roles of P-sel and TF-positive MPs and highlight strategies to modulate hemostasis by modulating the P-sel, TF, coagulation triad.Keywords: coagulation, microparticles, P-selectin, P-selectin glycoprotein ligand 1, thrombosis, tissue factor. P-selectin -link between inflammation and hemostasis P-selectin (P-sel) is a member of the selectin family of cell adhesion receptors, which mediate binding to specific carbohydrate-containing ligands [1]. Selectins mediate adhesion among leukocytes, platelets, and endothelium. P-sel, the largest member of the selectin family, is localized in the membranes of platelet a-granules [2] and in storage granules of endothelial cells called Weibel-Palade bodies [3,4] (Fig. 1). The major component of Weibel-Palade bodies is von Willebrand factor (VWF) [5]. VWF plays two main functions in hemostasis: it mediates platelet adhesion to the injured vessel wall, and it carries coagulation factor VIII. VWF directs the formation of the Weibel-Palade bodies and, in its absence, P-sel is mislocalized in the endothelial cell [6]. Weibel-Palade body exocytosis is triggered by secretagogues such as thrombin and histamine and occurs within seconds to minutes after stimulation. Similarly in platelets, upon activation, P-sel translocates with the a-granule membrane to the cell surface. Once present on the cell surface, the receptor can mediate cellular adhesion.A key role of P-sel is in mediating leukocyte interactions in inflammation [2]. P-sel has long been known to support leukocyte rolling, as shown in vitro [7] and in vivo [8]. The most clearly defined ligand for P-sel is P-selectin glycoprotein ligand 1 (PSGL-1). It is a homodimeric mucin expressed on the majority of leukocytes...

Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion

Plasma Factor XIII is a zymogen (plasma protransglutaminase) with the tetrametric structure A2B2, whereas the cellular protransglutaminase, i.e. Factor XIII in the platelet and monocyte/macrophage, consists exclusively of A subunits (A2). It is generally accepted that at Ca2+ concentrations comparable with that in plasma the proteolytic removal of an Nterminal activation peptide is the prerequisite for the Ca2+-induced formation of a catalytically active configuration of subunit A. In this study it was demonstrated that at high concentrations NaCl or KCI induced a non-proteolytic activation of cellular (placental macrophage) but not plasma protransglutaminase. The activation depended on time and salt concentration, and Ca2 , in the range 0-20 mm, greatly enhanced the activation process. At 1.25 M-NaCl maximal activation occurred within 60 min in the presence of 2 mM-CaC12, and even at physiological NaCl concentration a slow progressive activation could be observed in the presence of Ca2+. The specific activity of salt-activated Factor XIII was 1.5-2.0-fold higher than that obtained after thrombin activation. The non-proteolytic activation of cellular protransglutaminase was abolished by the addition of subunit B of plasma Factor XIII in stoichiometric amount, which suggests that (one of) the physiological function(s) of the B subunit in plasma Factor XIII is to prevent the slow spontaneous activation of A subunit that would occur in a plasmatic environment. INTRODUCTIONFactor XIII (FXIII) of blood coagulation present in the plasma is a zymogen (plasma protransglutaminase) of tetrameric structure (A2B2). The active site formed in the course of an activation process is located on the A subunit while subunit B remains enzymically inactive. The presence of FXIII has also been verified in platelets [1], monocytes and monocyte-derived macrophages [2][3][4][5][6][7]. In contrast with the plasma FXIII, however, this cellular protransglutaminase consists exclusively of A subunits (A2). The active transglutaminase (FXIIIa) formed from FXIII catalyses an acyl-transfer reaction in which the carboxamide group of a peptide-bound glutamine residue is the acyl