The role of platelet adhesion receptor GPIbalpha far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis.

GPIbalpha binding to von Willebrand factor (VWF) exposed at a site of vascular injury is thought to be the first step in the formation of a hemostatic plug. However, our previous studies in VWF-deficient mice demonstrated delayed but not absent arterial thrombus formation, suggesting that, under these conditions, GPIbalpha may bind other ligands or that a receptor other than GPIbalpha can mediate platelet adhesion. Here, we studied thrombus formation in transgenic mice expressing GPIbalpha in which the extracellular domain was replaced by that of the human IL-4 receptor (IL4Ralpha/GPIbalpha-tg mice). Platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4Ralpha/GPIbalpha-tg mice was virtually absent in contrast to avid adhesion in WT mice. As a consequence, arterial thrombus formation was inhibited completely in the mutant mice. Our studies further show that, when infused into WT recipient mice, IL4Ralpha/GPIbalpha-tg platelets or WT platelets lacking the 45-kDa N-terminal domain of GPIbalpha failed to incorporate into growing arterial thrombi, even if the platelets were activated before infusion. Surprisingly, platelets lacking beta3 integrins, which are unable to form thrombi on their own, incorporated efficiently into WT thrombi. Our studies provide in vivo evidence that GPIbalpha absolutely is required for recruitment of platelets to both exposed subendothelium and thrombi under arterial flow conditions. Thus, GPIbalpha contributes to arterial thrombosis by important adhesion mechanisms independent of the binding to VWF.

Decreased plasma fibronectin leads to delayed thrombus growth in injured arterioles.

OBJECTIVE: Plasma fibronectin (FN) is decreased in several clinical conditions. We were interested to study the thrombotic and hemostatic consequences of the decrease in plasma FN (pFN), the role of FN splice variants in thrombosis, and to examine whether pFN incorporates into thrombi in vivo. METHODS AND RESULTS: We compared the thrombotic response to a vessel injury in FN heterozygous (FN+/-) mice and corresponding FN+/+ mice. Although normal thrombosis in venules was observed, a decrease to half in the pFN concentration in FN+/- mice caused a delay in the appearance of thrombi in arterioles and consequently a delay in their occlusion. We were able to rescue the thrombotic defect in the FN+/- mice by infusion of rat pFN. Additionally, we could show intense incorporation of fluorescent pFN-coated microspheres into the developing thrombi. Moreover, we found that mice expressing FN without the EIIIA or EIIIB domains specific to cellular FN including platelet FN had no thrombotic defect. CONCLUSIONS: Mice heterozygous for FN have a striking defect in thrombus initiation and growth in arterioles attributable to the decrease of pFN. Our study is an example of haploid insufficiency for FN, and it emphasizes the fundamental role of this plasma protein in thrombosis in the arterial system.

Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo.

Interaction of the platelet receptor glycoprotein (GP) Ib-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIbalpha subunit was first described >25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We reported recently that shedding of GPIbalpha induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIbalpha shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-alpha converting enzyme (TACE(DeltaZn/DeltaZn)) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of 2 potent TACE inhibitors: TAP1 and TMI-1. Our data further suggest that TACE is important in the regulation of GPIbalpha expression in vivo because we observed an approximately 90% reduction in soluble GPIbalpha (glycocalicin) in plasma of TACE(DeltaZn/DeltaZn) chimeras as well as significantly increased levels of GPIbalpha on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Damaged TACE(DeltaZn/DeltaZn) platelets were further characterized by a markedly improved post-transfusion recovery and hemostatic function in mice. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIbalpha.

CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation.

Signaling through the second messengers calcium and diacylglycerol (DAG) is a critical element in many biological systems. Integration of calcium and DAG signals has been suggested to occur primarily through protein kinase C family members, which bind both calcium and DAG. However, an alternative pathway may involve members of the CalDAG-GEF/RasGRP protein family, which have structural features (calcium-binding EF hands and DAG-binding C1 domains) that suggest they can function in calcium and DAG signal integration. To gain insight into the signaling systems that may be regulated by CalDAG-GEF/RasGRP family members, we have focused on CalDAG-GEFI, which is expressed preferentially in the brain and blood. Through genetic ablation in the mouse, we have found that CalDAG-GEFI is crucial for signal integration in platelets. Mouse platelets that lack CalDAG-GEFI are severely compromised in integrin-dependent aggregation as a consequence of their inability to signal through CalDAG-GEFI to its target, the small GTPase Rap1. These results suggest that analogous signaling defects are likely to occur in the central nervous system when CalDAG-GEFI is absent or compromised in function.

GPVI down-regulation in murine platelets through metalloproteinase-dependent shedding.

Platelet interaction with subendothelial collagen is crucial for hemostasis and thrombosis. Under conditions of elevated shear, platelet adhesion and activation on collagen requires the coordinated action of glycoprotein (GP) Ib and GPVI, which may be physically and functionally linked in the platelet membrane. While the surface expression of GPIb can be down-regulated by internalization and/or proteolytic cleavage of a 130 kDa fragment of GPIb (glycocalicin, GC), very little is known about the cellular regulation of GPVI. We have recently shown that GPIb on platelets is cleaved by metalloproteinase-dependent mechanisms in response to mitochondrial injury. In the current study, we examined a possible role of platelet metalloproteinases in the regulation of GPVI. Mitochondrial injury induced by incubation of mouse platelets with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) severely affected the cells' responses to collagen or the GPVI-specific agonist, collagen related peptide (CRP), but not to thrombin or the stable thromboxane A(2) analog U46619. This defect was due to a rapid proteolytic cleavage of GPVI, as shown by the release of the 55 kDa extracellular domain into the supernatant. Both the proteolysis of GPVI and the loss of its activity were inhibited in the presence of the broad range metalloproteinase inhibitor, GM6001. Platelet stimulation with thrombin or CRP, however, resulted in marked metalloproteinase-dependent shedding of GPIbalpha, but not GPVI suggesting that different metalloproteinases are involved in the regulation of the two receptors or, alternatively, an additional signal is required to render GPVI susceptible to cleavage.

Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro-aged or -injured mouse platelets.

Platelet transfusions are a crucial component of support for patients with severe thrombocytopenia. Storage of platelet concentrates, however, is associated with a reduction in platelet posttransfusion recovery and hemostatic function. In this study, we established a model of mitochondrial injury that resembles platelet storage lesion. Mitochondrial injury, provoked by incubation of platelets with carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to reduced posttransfusion recovery in mice, an effect that directly correlated with the duration of treatment. Damaged platelets were characterized by shape change, disruption of membrane asymmetry, surface expression of P-selectin, and profound proteolysis of GPIbalpha. Using our model, we identified a key role for endogenous metalloproteinase(s) in platelet clearance, as their inhibition markedly improved posttransfusion recovery of both the mitochondria-injured and in vitro-aged mouse platelets. Metalloproteinase inhibition also prevented proteolysis of GPIbalpha on damaged platelets, thereby improving the hemostatic function of these cells in vivo. We propose that inhibition of metalloproteinase activity during storage could significantly improve the effectiveness of platelet transfusions. Surface expression of GPIbalpha might be a powerful marker to determine the quality of platelet concentrates, because it reflects metalloproteinase activity in vitro.