Deep vein thrombosis initiation is mediated by cross talk between monocytes, neutrophils, and platelets.

In addition to their pivotal role in thrombosis and wound repair, platelets participate in inflammatory responses. We investigated the role of platelets in the autoimmune disease rheumatoid arthritis. We identified platelet microparticles—submicrometer vesicles elaborated by activated platelets—in joint fluid from patients with rheumatoid arthritis and other forms of inflammatory arthritis, but not in joint fluid from patients with osteoarthritis. Platelet microparticles were proinflammatory, eliciting cytokine responses from synovial fibroblasts via interleukin-1. Consistent with these findings, depletion of platelets attenuated murine inflammatory arthritis. Using both pharmacologic and genetic approaches, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. Thus, these findings demonstrate a previously unappreciated role for platelets and their activation-induced microparticles in inflammatory joint diseases.

Although once primarily recognized for its roles in hemostasis and thrombosis, the platelet has been increasingly recognized as a multipurpose cell. Indeed, circulating platelets have the ability to influence a wide range of seemingly unrelated pathophysiologic events. Here, we highlight some of the notable observations that link platelets to inflammation, reinforcing the platelet’s origin from a lower vertebrate cell type with both hemostatic and immunologic roles. In addition, we consider the relevance of platelets in cancer biology by focusing on the hallmarks of cancer and the ways platelets can influence multistep development of tumors. Beyond its traditional role in hemostasis and thrombosis, the platelet’s involvement in the interplay between hemostasis, thrombosis, inflammation, and cancer is likely complex, yet extremely important in each disease process. The existence of animal models of platelet dysfunction and currently used antiplatelet therapies provide a framework for understanding mechanistic insights into a wide range of pathophysiologic events. Thus, the basic scientist studying platelet function can think beyond the traditional hemostasis and thrombosis paradigms, while the practicing hematologist must appreciate platelet relevance in a wide range of disease processes.

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Proteolytic cleavage of the von Willebrand factor subunit may be important for processing and/or function of the molecule and is altered in certain subtypes of von Willebrand disease. It results in the generation of two main fragments with apparent molecular masses of 140 kDa and 176 kDa from the 225-kDa subunit. We have now obtained chemical evidence to locate the protease-sensitive bond between residues Tyr-842 and Met-843, a site that appears to reflect the specificity of calcium-dependent neutral proteases (calpains). Antibodies were raised against four synthetic peptides that represented sequences immediately preceding or following or including the cleavage site. One antibody (against the fragment from Ala-837 through Asp-851) reacted only with the intact subunit, and its epitope included the cleavage site. All others reacted specifically with either the 140-kDa or the 176-kDa fragment, demonstrating their origin from a single cleavage. In samples of purified von Willebrand factor from four of five patients with type HA von Willebrand disease, the anti-peptide antibodies showed markedly decreased reactivity with either the 140-kDa or the 176-kDa fragment, suggesting the existence of distinct molecular abnormalities clustered around the cleavage site. Thus, in the majority of type HA patients, a common pathogenetic mechanism may lead to the disappearance of the larger multimers as a consequence of structural changes that may expose a sensitive bond to the action of specific proteases. These studies demonstrate the use of anti-peptide antibodies directed at a relevant structural domain for the immunochemical differentiation of normal and mutant molecules.

The human Bernard-Soulier syndrome is an autosomal recessive disorder of platelet dysfunction presenting with mild thrombocytopenia, circulating ''giant'' platelets and a bleeding phenotype. The bleeding in patients with the Bernard-Soulier syndrome is disproportionately more severe than suggested by the reduced platelet count and is explained by a defect in primary hemostasis owing to the absence of the platelet glycoprotein (GP) Ib-IX-V membrane receptor. However, the molecular basis for the giant platelet phenotype and thrombocytopenia have remained unresolved but assumed to be linked to an absent receptor complex. We have disrupted the gene encoding the ␣-subunit of mouse GP Ib-IX-V (GP Ib␣) and describe a murine model recapitulating the hallmark characteristics of the human Bernard-Soulier syndrome. The results demonstrate a direct link between expression of a GP Ib-IX-V complex and normal megakaryocytopoiesis and platelet morphogenesis. Moreover, using transgenic technology the murine Bernard-Soulier phenotype was rescued by expression of a human GP Ib␣ subunit on the surface of circulating mouse platelets. Thus, an in vivo model is defined for analysis of the human GP Ib-IX-V receptor and its role in the processes performed exclusively by megakaryocytes and platelets.

Platelets contain both pro-and antiangiogenic factors, but their regulatory role in angiogenesis is poorly understood. Although previous studies showed that platelets stimulate angiogenesis in vitro, the role of platelets in angiogenesis in vivo is largely uncharacterized. To address this topic, we used two in vivo approaches, the cornea micropocket assay and the Matrigel model, in four animal models: thrombocytopenic, Lyst bg (platelet storage pool deficiency), glycoprotein (GP) Ib␣͞IL4R transgenic (lacking extracellular GPIb␣, the receptor for von Willebrand factor as well as other adhesive and procoagulant proteins), and Fc␥R ؊/؊ (lacking functional GPVI, the collagen receptor) mice. Adult mice were rendered thrombocytopenic by i.p. administration of an antiplatelet antibody. The number of growing vessels in the thrombocytopenic mice was lower in the cornea assay, and they showed significantly increased appearance of hemorrhage compared with mice treated with control IgG. The thrombocytopenic mice also showed more protein leakage and developed hematomas in the Matrigel model. GPIb␣͞IL4R transgenic mice presented increased hemorrhage in both assays, but it was less severe than in the platelet-depleted mice. Fc␥R ؊/؊ and Lyst bg mice showed no defect in experimental angiogenesis. Intravital microscopy revealed a >3-fold increase in platelet adhesion to angiogenic vessels of Matrigel compared with mature quiescent skin vessels. Our results suggest that the presence of platelets not only stimulates angiogenic vessel growth but also plays a critical role in preventing hemorrhage from the angiogenic vessels. The adhesion function of platelets, as mediated by GPIb␣, significantly contributes to the process. thrombocytopenic mice ͉ blood vessel ͉ ␣-granule ͉ cornea ͉ collagen receptor

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