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Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.

In addition to their hemostatic function, platelets play an important role in regulating the inflammatory response. The platelet NLRP3 inflammasome not only promotes interleukin-1β secretion, but was also found to be upregulated during platelet activation and thrombus formation in vitro. However, the role of NLRP3 in platelet function and thrombus formation in vivo remains unclear. In this study, we aimed to investigate the role of NLRP3 in platelet integrin αIIbβ3 signaling transduction. Using NLRP3−/− mice, we showed that NLRP3-deficient platelets do not have significant differences in expression of the platelet-specific adhesive receptors αIIbβ3 integrin, GPIba or GPVI; however, NLRP3−/− platelets transfused into wild-type mice resulted in prolonged tail-bleeding time and delayed arterial thrombus formation, as well as exhibiting impaired spreading on immobilized fibrinogen and defective clot retraction, concomitant with decreased phosphorylation of c-Src, Syk and PLCγ2 in response to thrombin stimulation. Interestingly, addition of exogenous recombinant interleukin-1β reversed the defect in NLRP3−/− platelet spreading and clot retraction, and restored thrombin-induced phosphorylation of c-Src/Syk/PLCγ2, whereas an anti-interleukin-1β antibody blocked spreading and clot retraction mediated by wild-type platelets. Using the direct NLRP3 inhibitor, CY-09, we demonstrated significantly reduced human platelet aggregation in response to threshold concentrations of collagen and ADP, as well as impaired clot retraction in CY-09-treated human platelets, supporting a role for NLRP3 also in regulating human platelet αIIbβ3 outside-in signaling. This study identifies a novel role for NLRP3 and interleukin-1β in platelet function, and provides a new potential link between thrombosis and inflammation, suggesting that therapies targeting NLRP3 or interleukin-1β might be beneficial for treating inflammation-associated thrombosis.

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Gene targeting was used to evaluate the physiological function of mouse calpain-1 and establish that its inactivation results in reduced platelet aggregation and clot retraction potentially by causing dephosphorylation of platelet proteins. Here, we report that calpain-1 null (Capn1−/−) platelets accumulate protein tyrosine phosphatase 1B (PTP1B), which correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates. Treatment of Capn1−/− platelets with bis(N,N-dimethylhydroxamido)hydroxooxovanadate, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. More importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Further evaluation of mutant mice by the ferric chloride-induced arterial injury model suggests that the Capn1−/− mice are relatively resistant to thrombosis in vivo. Together, our results demonstrate that PTP1B is a physiological target of calpain-1 and suggest that a similar mechanism may regulate calpain-1-mediated tyrosine dephosphorylation in other cells.

Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, α2-plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. In contrast, there was no CR impairment in the PRP of tissue transglutaminase-knockout mice compared with that of wild-type mice. Furthermore, a transglutaminase inhibitor, cystamine, halted CR in the PRP of wild-type mice. These results indicate that the enzymatic activity of FXIII is necessary for CR, at least in mice.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Previously we used gene-targeting to evaluate the physiological function of mouse calpain-1, and established that its inactivation results in reduced platelet aggregation and clot retraction, potentially by causing dephosphorylation of platelet proteins. Here, we present data showing that calpain-1 null platelets accumulate protein tyrosine phosphatase 1B (PTP1B) that correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates in platelets. Using antibodies specific for phosphotyrosines 747 and 759 of the b3 subunit of αIibβ3 integrin, we show that the tyrosine phosphorylation of both tyrosine residues at positions 747 and 759 in the cytoplasmic domain of b3 subunit is reduced by approximately 60–70% in the calpain-1 null platelets. Treatment of calpain-1 null platelets with DMHV, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. Importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Consistent with this paradigm, treatment of wild type mouse platelets as well as human platelets with the tyrosine phosphatase inhibitor DMHV enhanced their aggregation at low doses of thrombin. Conversely, MDL, a cell permeable inhibitor of calpains, potently inhibited aggregation of wild type mouse platelets in a dose-dependent manner upon thrombin activation. Further evaluation of mutant mice by ferric chloride induced arterial injury model suggests that the calpain-1 null mice are relatively resistant to thrombosis in vivo. Finally, the calpain-1 mediated regulation of PTP1B appears to be a systemic event as evident by the enhanced tyrosine dephosphorylation of B lymphocytes and their resistance to apoptosis in calpain-1 null mice. Together, our results demonstrate that PTP1B is a physiological substrate of calpain-1 and suggest that a similar mechanism may regulate calpain-1 mediated tyrosine dephosphorylation in other cells.

A case of von Willebrand’s disease is presented. Three sibs died from haemorrhage and two living sibs have negative findings. Epistaxis, purpura and prolonged haemorrhage on trauma are the symptoms. Prolonged bleeding time, impaired clot retraction, abnormal nailbed capillary morphology and injury-reaction are the findings. Aetiology and treatment are discussed.

We have described a case of thrombocytopenic purpura caused by methyldopa. A methyldopa-dependent antiplatelet antibody as the mechanism of the patient's thrombopenia was suggested by a positive clot retraction inhibition test. This simple in vitro test should be done more often in suspected cases of drug-induced thrombocytopenia.

SummaryA specific complement-reactive factor derived from cobra venom (CVF) inhibited complement activity, impaired clot retraction, and induced aggregation and release of intracellular constituents in plateletrich plasma or suspensions of washed platelets from guinea pigs, rabbits, dogs, and humans. Platelets from thrombopathic dogs and from a complement deficient (C3) patient responded similarly when mixed with purified CVF; possible explanations for these observations are included.We are indebted to Mr. Jack Newman, New York University Medical Center, for testing our CVF preparations for the presence of contaminating fibrinolytic enzymes.

IDIOPATHIC thrombopenic purpura is a symptom complex characterized by petechial hemorrhages, thrombopenia, impaired clot retraction and prolonged bleeding time. Widespread internal hemorrhages may occur and cause the death of the patient. The symptom complex is frequently encountered, but the more seriously affected patients are comparatively rare. Although Werlhof1is generally considered as having described this disease first, in 1775, it was Brohm2who, in 1883, first observed thrombopenia in a case of purpuric disease. It has been assumed that Werlhof's patient had thrombopenia; hence, the disease bears his name. Interest has centered chiefly on the role of blood platelets in this disease. The existence of platelets was recognized by several workers in the early nineteenth century, but for many years their existence as real or as artificial blood elements was in controversy. It was the result of the collective researches of Zimmerman,3Schultze,4Osler,5Bizzozero