Properties of soluble glycoprotein VI, a potential platelet activation biomarker.

Glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. Clinical studies suggest that the plasma level of soluble GPVI (sGPVI) is a highly specific and useful platelet activation marker. However, many properties of sGPVI have not been fully characterized, such as its sensitivity in detecting platelet activation and its elimination rate from the blood. In this study we established a sandwich enzyme-linked immunosorbent assay for human sGPVI, which cross-reacts to cynomolgus monkey sGPVI, and evaluated the time course of sGPVI production in a cynomolgus monkey model of lipopolysaccharide (LPS)-induced thrombocytopenia. The sGPVI levels in this model were dramatically elevated and returned to baseline by 24 hours after LPS injection, the change was more pronounced than the existing platelet activation biomarker, soluble P-selectin (sP-selectin) levels. The elimination half-life of recombinant human sGPVI was about 2.5 hours following intravenous administration to monkeys. These results suggest that plasma sGPVI closely reflects platelet activation in the bloodstream and has a short half-life. sGPVI would be a useful biomarker for disorders marked by platelet activation and for monitoring anti-platelet therapy.

Phagocytosis by human monocytes is required for the secretion of presepsin.

BACKGROUND: Presepsin, a soluble CD14 subtype, is increasingly recognized as a useful biomarker for sepsis. However, little is known about the biological characteristics of presepsin in humans. Furthermore, there are no studies evaluating clinical validity of measuring the presepsin levels in patients after allogeneic hematopoietic cell transplantation, irrespective of the high frequency of sepsis. METHODS: For in vitro assays, neutrophils and monocytes were isolated from the peripheral blood of healthy controls and treated with bacteria or inflammatory stimuli. Presepsin levels in the culture supernatants were measured by enzyme linked immunosorbent assay (ELISA). For a cohort study of patients undergoing allogeneic hematopoietic cell transplantation, serum samples were subjected to ELISA for presepsin, and the relationship of presepsin levels with the incidence of transplantation-related complications was statistically analyzed. RESULTS: We found that monocytes were the main source of presepsin in humans. Presepsin secretion by human monocytes was triggered by bacterial phagocytosis or sterile phagocytic stimulus, such as monosodium urate crystals, rather than soluble inflammatory stimuli. Elastase, a serine protease in human monocytes, mediated CD14 cleavage to produce presepsin. The cohort study demonstrated that high presepsin values were significantly associated with an increased incidence of hemophagocytic syndrome, as well as bacteremia. Moreover, patients with higher presepsin values revealed inferior overall survival, suggesting that presepsin can also be a prognostic marker for transplantation. CONCLUSIONS: In this study, we clarified the biological features of presepsin in humans. Our study may be useful for increasing the clinical application of presepsin as a biomarker.

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain complex.

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

Elevated Soluble Platelet Glycoprotein VI Levels in Patients After Living Donor Liver Transplantation.

Plasma-soluble platelet glycoprotein VI (sGPVI) levels were examined in patients undergoing living donor liver transplantation (LDLT), and the relationship between platelet activation and thrombocytopenia was evaluated to understand the mechanism of thrombocytopenia in LDLT. Platelet counts were significantly higher in the donors compared to the recipient, and the plasma sGPVI levels increased in both groups after the operation. Regarding the relationship between the platelet counts and the sGPVI levels, the slope varied on different days, and it became negative on day 3, suggesting that the plasma sGPVI levels are related to platelet activation in LDLT. The frequency of complications was high in the nonsurvivors. The platelet counts were higher in the survivors than in the nonsurvivors on days 14 and 28. Although the plasma levels of sGPVI in the survivors increased after the operation, those in the nonsurvivors were high only on day 3. Although the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 domain, member 13) levels were markedly reduced, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were markedly elevated during LDLT. The antithrombin activity was significantly lower (day 14) and VWFpp (day 28) was significantly higher in the nonsurvivors than in the survivors. These findings suggest that platelet activation first occurs after LDLT, and it is high in the nonsurvivors on day 3. Thereafter, the hemostatic abnormality and vascular endothelial cell injuries may appear on days 14 and 28.

Establishment of immunoassay for platelet-derived soluble glycoprotein VI, a novel platelet marker.

Soluble Glycoprotein VI (GPVI) is an attractive marker for disorders marked by platelet activation, such as thrombotic microangiopathy, myocardial infarction, and stroke. Several groups have already developed an immunoassay for soluble GPVI; however, there are several discrepancies between the groups' assays. In this study, we prepared the two types of recombinant soluble GPVI, the monomeric form GPVI (GPVI-His) and the dimeric form of GPVI (GPVI-Fc), moreover, we generated four anti-GPVI antibodies, F1232-7-1 (7S1), F1232-10-2 (10S2), F1232-19-1 (19D1), and F1232-21-1 (21D1). The former 2 antibodies (7S1 and 10S2) had a high affinity for both GPVI-His and GPVI-Fc, while the latter 2 antibodies (19D1 and 21D1) showed a high affinity for GPVI-Fc but low affinity for GPVI-His. All of the antibodies comparably recognized surface GPVI on resting platelets. Furthermore, we established two immunoassays for soluble GPVI, 7S1/10S2-HRP and 19D1/21D1-HRP (capture antibody/detection antibody). 7S1/10S2-HRP showed equivalent reactivity with GPVI-His and GPVI-Fc, whereas 19D1/21D1-HRP had high affinity for GPVI-Fc but low reactivity with GPVI-His. In terms of reactivity with platelet-derived soluble GPVI, 7S1/10S2-HRP demonstrated sensitive detection whereas 19D1/21D1-HRP was nonreactive. Taken together, 7S1/10S2-HRP is a suitable candidate for a reliable soluble GPVI immunoassay as it has a high affinity for monomeric GPVI.

Elevated plasma levels of soluble platelet glycoprotein VI (GPVI) in patients with thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets. This study examined the soluble platelet glycoprotein VI (sGPVI) levels in patients with TMA to evaluate the activation of platelets in thrombotic states. MATERIALS AND METHODS: The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in patients with TMA. RESULTS: The plasma levels of sGPVI were significantly higher in postoperative patients, patients with TMA and those with disseminated intravascular coagulation (DIC) than in those without thrombosis. The plasma levels of sGPVI were the highest in patients with TMA without markedly reduced ADAMTS13 and those were significantly reduced after plasma exchange. CONCLUSION: The measurement of sGPVI level is therefore considered to be important for the diagnosis and evaluation of TMA.

Elevated soluble platelet glycoprotein VI is a useful marker for DVT in postoperative patients treated with edoxaban.

Prevention of deep vein thrombosis (DVT) is important in patients undergoing major orthopedic surgery. Although the detection of an elevated D-dimer level is useful for predicting DVT, it is not efficacious in postoperative patients being treated with anti-Xa agents. The soluble platelet glycoprotein VI (sGPVI) level is a marker of activated platelets, but not bleeding. Therefore, sGPVI levels are usually examined as a predictor of DVT in such patients. In the present study, 83 orthopedic patients were treated with 30 mg of edoxaban for prophylaxis of DVT. Fourteen patients developed DVT and 17 patients discontinued the prophylaxis due to decreased hemoglobin levels. Plasma levels of sGPVI in the patients were significantly higher after surgery than before surgery. On day 1, the sGPVI levels increased, while the platelet counts decreased. There were no significant differences in D-dimer, soluble fibrin, or FDP levels in orthopedic patients with and without DVT before surgery and on days 1, 4, and 8. Plasma sGPVI levels were significantly higher in the patients with DVT than in those without DVT on days 1 and 4. Plasma levels of D-dimer were significantly higher in patients with withdrawal than in those without. However, there were no significant differences in sGPVI levels between those with and without withdrawal. As D-dimer levels are known to increase in patients with withdrawal, this parameter is not useful for evaluating the risk of DVT in these patients. In contrast, the sGPVI level is not increased in those with withdrawal and may therefore be useful for evaluating the risk of DVT in postoperative patients treated with an anticoagulant.