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Introduction: Diabetes mellitus (DM) is a prothrombotic and proinflammatory state. Hyperglycemia (HG) is encountered even in patients without DM. We have shown that combined HG and hyperinsulinemia (HI) in healthy non-diabetic subjects increased circulating tissue factor (TF) and thrombin generation. To understand the changes in platelet and monocyte pathways induced by combined HG and HI in healthy non-diabetic state, we performed whole genome expression profiling of leukocyte-depleted platelets and monocytes before and after 24 hours of combined HG (glucose ~200mg/dL) and HI by glucose infusion clamp in a healthy non-diabetic subject., Results: We defined time-dependent differential mRNA expression (24 versus 0 hour fold change (FC) ≥ 2) common to platelets and monocytes. Ingenuity Pathways Analysis revealed alterations in canonical insulin receptor signaling and coagulation pathways. A preliminary group of 9 differentially expressed genes was selected for qRT-PCR confirmation. Platelet 24 hour sample was compared to the 0 hour sample plus 4 controls. Five transcripts in platelets and 6 in monocytes were confirmed. Platelet GSK3B and PTPN1 were upregulated, and STXBP4 was downregulated in insulin signaling, and F3 and TFPI were upregulated in coagulation pathways. Monocyte, PIK3C3, PTPN11 and TFPI were downregulated. Platelet GSKβ3 and PTPN11 protein and TF antigen in platelets and monocytes was increased., Conclusions: Even in non-diabetic state, HG+HI for 24 hours induces changes in platelets and monocytes. They suggest downregulation of insulin signaling and upregulation of TF. Further studies are needed to elucidate cellular alterations leading to the prothrombotic and proinflammatory state in DM., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.

Congenital defects in platelet function are associated with bleeding manifestations of variable intensity and arise by diverse mechanisms. Defects in platelet-vessel wall interaction (disorders of adhesion) may arise because of qualitative or quantitative abnormalities in plasma von Willebrand factor (von Willebrand disease) or in platelet glycoprotein Ib, the binding site on platelets for von Willebrand factor (Bernard-Soulier syndrome). Disorders characterized by abnormal platelet-platelet interaction (disorders of aggregation) arise because of absence of plasma fibrinogen (congenital afibrinogenemia) or because of qualitative or quantitative abnormalities in platelet glycoprotein IIb-IIIa complex (Glanzmann's thrombasthenia). Patients with abnormalities in platelet secretion and signal transduction are a heterogeneous group characterized by impaired aggregation responses and secretion of granule contents. A small proportion of these patients have deficiency of granule stores (storage pool deficiency [SPD]) or impaired production of thromboxane A2; in most, the mechanisms underlying the platelet dysfunction are unknown. Evidence is accumulating that at least some patients have abnormalities in early signal transduction events. Abnormalities in phospholipase C activation, G-protein activation, and other events (eg, protein phosphorylation) have been documented. Platelets play a major role in blood coagulation, and in Scott syndrome, there is an abnormality in platelet contribution to the mechanisms leading to thrombin generation. In most patients with inherited platelet dysfunction, the underlying mechanisms remain to be delineated. Future studies of these patients should yield valuable new information on normal platelet mechanisms.

Defects in signal transduction mechanisms may underlie the impaired aggregation and secretion in patients with congenital platelet function defects (CPD). Both protein kinase C (PKC) induced pleckstrin phosphorylation and cytoplasmic Ca2+ mobilization play a major role in secretion. We postulated that combined platelet activation with a cell permeable direct PKC activator 1,2-dioctanoyl-sn-glycerol (DiC8) and ionophore A23187, which possibly bypass the steps involved in the intracellular synthesis of two major mediators (inositol trisphosphate, diacylglycerol), may induce normal dense granule secretion in patients with impaired receptor mediated secretion. We studied eight CPD patients with abnormal aggregation and secretion in response to several different surface receptor-mediated agonists despite the presence of normal dense granule contents. Receptor mediated Ca2+ mobilization and/or pleckstrin phosphorylation were abnormal in seven patients. Platelet activation with a combination of ADP (8 microM) with DiC8 (200 microM) or A23187 (10 microM) improved secretion in four patients. However, platelet activation with a combination of 200 microM DiC8 with 10 microM A23187, or 100 microM DiC8 with 5 microM A23187 induced normal secretion in platelet-rich plasma in all patients. These studies suggest that in such patients with CPD the ultimate process of exocytosis or secretion per se is intact and impaired secretion results from abnormalities in early signal transduction events, possibly upstream of diacylglycerol formation and calcium mobilization. Detailed studies are needed to delineate the specific abnormalities in these heterogenous patients with signal transduction defects.

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.

Signal transduction on platelet activation involves phosphoinositide-specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet-activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate-induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.