Hemostasis and pathological thrombus formation are dynamic processes that require a co-ordinated series of events involving platelet membrane receptors, bidirectional intracellular signals, and release of platelet proteins and inflammatory substances. This review aims to summarize current knowledge in the key steps in the dynamics of thrombus formation, with special emphasis on the crucial participation of platelet receptors and signaling in this process. Initial tethering and firm adhesion of platelets to the exposed subendothelium is mediated by glycoprotein (GP) Ib/IX/V complex and collagen receptors, GP VI and α₂β₁ integrin, in the platelet surface, and by VWF and fibrillar collagen in the vascular site. Interactions between these elements are largely influenced by flow and trigger signaling events that reinforce adhesion and promote platelet activation. Thereafter, soluble agonists, ADP, thrombin, TxA₂, produced/released at the site of vascular injury act in autocrine and paracrine mode to amplify platelet activation and to recruit circulating platelets to the developing thrombus. Specific interactions of these agonists with their G-protein coupled receptors generate inside-out signaling leading to conformational activation of integrins, in particular α_IIbβ₃, increasing their ligand affinity. Binding of α_IIbβ₃ to its ligands, mainly fibrinogen, supports processes such as clot retraction and platelet aggregation. Stabilization of thrombi is supported by the late wave of signaling events promoted by close contact between aggregated platelets. The best known contact-dependent signaling is outside-in signaling through αIb β₃, but new ones are being clarified such as those mediated by interaction of Eph receptors with ephrins, or by Sema 4D and Gas-6 binding to their receptors. Finally, newly identified mechanisms appear to control thrombus growth, including back-shifting of activated integrins and actuation of compensatory molecules such as ESAM or PECAM-1. The expanding knowledge of thrombotic disease is expected to translate into the development of new drugs to help management and prevention of thrombosis.

Inherited thrombocytopenias are a heterogeneous group of disorders characterised by abnormally low platelet counts which can be associated with abnormal bleeding. Next generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease causing genes. However its full potential has not previously been utilised. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown aetiology with platelet counts varying from 11-186x109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified pathogenic or likely pathogenic variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases which include novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventive treatments. Until recently, this has been performed by Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of disorder. The remaining had disease with no specific manifestations. Overall, high-throughput sequencing test attained a molecular diagnosis in 70% of the patients. This sensitivity increased to 90% among patients suspected to have a defined platelet disorder. We found 58 different candidate variants in 29 genes, among which 70% were not previously described. Following consensus guidelines, we qualified 68% and 27% of the candidate variants as pathogenic and likely pathogenic, respectively. Apart from establishing definitive diagnosis of well-known inherited platelet disorders in most patients, in few others high-throughput sequencing provided molecular results suggestive of less recognized disorders, such as sitosterolemia, filamin and actinin deficiencies and abnormalities at the level of G-protein coupled receptors and effector enzymes. Noteworthy, high-throughput sequencing diagnosed novel patients with very rare molecular pathology in DIAPH1 (n=2) and RASGRP2 (n=3). Overall, our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream of genetic diagnosis practice in inherited platelet disorders.

Platelet membrane glycoprotein Ib (GPIb) is a major receptor for von Willebrand factor and thrombin, which plays a key role in the initial development of thrombi. Two polymorphisms (HPA-2 and VNTR) that affect phenotype have been described in GPIb. The relevance of these polymorphisms to thrombotic disease was investigated by genotypic identification in three case-control studies: 104 case patients with acute cerebrovascular disease (CVD), 101 case patients with acute coronary heart disease (CHD), 95 patients with deep venous thrombosis (DVT), and one control age-, sex-, and race-matched for each case patient. Results show that the C/B genotype of the VNTR and the HPA-2b polymorphisms of GPIb are strongly associated with increased risk of coronary heart disease and cerebral vascular disease but not with deep vein thrombosis. These two polymorphisms of GPIb may represent newly identified risk factors for arterial thrombotic disease, but not for venous thrombosis.© 1998 by The American Society of Hematology.

Platelets play a major role in hemostasis as ppwell as in many other physiological and pathological processes. Accordingly, production of about 10¹¹ platelet per day as well as appropriate survival and functions are life essential events. Inherited platelet disorders (IPDs), affecting either platelet count or platelet functions, comprise a heterogenous group of about sixty rare diseases caused by molecular anomalies in many culprit genes. Their clinical relevance is highly variable according to the specific disease and even within the same type, ranging from almost negligible to life-threatening. Mucocutaneous bleeding diathesis (epistaxis, gum bleeding, purpura, menorrhagia), but also multisystemic disorders and/or malignancy comprise the clinical spectrum of IPDs. The early and accurate diagnosis of IPDs and a close patient medical follow-up is of great importance. A genotype–phenotype relationship in many IPDs makes a molecular diagnosis especially relevant to proper clinical management. Genetic diagnosis of IPDs has been greatly facilitated by the introduction of high throughput sequencing (HTS) techniques into mainstream investigation practice in these diseases. However, there are still unsolved ethical concerns on general genetic investigations. Patients should be informed and comprehend the potential implications of their genetic analysis. Unlike the progress in diagnosis, there have been no major advances in the clinical management of IPDs. Educational and preventive measures, few hemostatic drugs, platelet transfusions, thrombopoietin receptor agonists, and in life-threatening IPDs, allogeneic hematopoietic stem cell transplantation are therapeutic possibilities. Gene therapy may be a future option. Regular follow-up by a specialized hematology service with multidisciplinary support especially for syndromic IPDs is mandatory.