Hiroyuki Takeya

Protein C activation in NIDDM patients

Summary Enhanced activation of the clotting system has been recently implicated in the pathogenesis of vascular complications in patients with diabetes mellitus. Abnormalities of the anticoagulant system may constitute a potential trigger factor for the haemostatic activation observed in diabetic subjects. The current study aimed to evaluate anticoagulant activity in diabetic patients by assessing the plasma levels of activated protein C-protein C inhibitor complex; and by measuring the anticoagulant response to exogenous thrombomodulin. This study comprised 61 patients (34 men, 27 women) with non-insulin-dependent diabetes mellitus (NIDDM) of whom 22 showed microalbuminuria and 39 normoalbuminuria. Data obtained in 31 non-obese and non-diabetic subjects were available for comparison. The plasma levels of fibrinogen (p < 0.02), prothrombin fragment 1 + 2 (p < 0.05), fibrin monomer (p < 0.0001), protein C antigen (p < 0.005), total protein S antigen (p < 0.02), soluble thrombomodulin (p < 0.005) and soluble E-selectin (p < 0.005) were significantly higher in diabetic patients than in healthy subjects. The plasma level of activated protein C-protein C inhibitor complex (7.4 ± 3.8 vs 3.0 ± 0.4 pmol/l) was significantly higher (p < 0.0001) and the anticoagulant response to exogenous thrombomodulin (23.4 ± 2.6 vs 35.3 ± 3.0 ng/ml) was markedly lower (p = 0.005) in all diabetic patients than in healthy subjects. Cases with microalbuminuria presented low plasma levels of activated protein C-protein C inhibitor complex (5.5 ± 0.6 vs 8.6 ± 0.7 pmol/l, p < 0.05) and significantly decreased values of the anticoagulant response to exogenous thrombomodulin (16.5 ± 2.9 vs 23.4 ± 2.6 %, p = 0.03) as compared to those with normoalbuminuria. The present study suggests that the hyper-coagulable state in NIDDM is associated with an increased activation of protein C but with a poor plasma reactivity to the anticoagulant effect of thrombomodulin. [Diabetologia (1996) 39: 1455–1461]

Changes of plasma hemostatic markers during percutaneous transluminal coronary angioplasty in patients with chronic coronary artery disease

Changes of hemostatic parameters during percutaneous transluminal coronary angioplasty (PTCA) in 75 patients with chronic coronary artery disease were evaluated. Plasma levels of D‐dimer, soluble fibrin monomer, plasmin‐α2 antiplasmin inhibitor complex, and tissue factor (TF) were significantly increased in all patients with chronic coronary artery disease. The activity of antithrombin and protein C and the levels of protein C antigen were significantly decreased 1 hr after PTCA, but they returned to normal range 1 day after PTCA. There was no significant difference in the level of plasma APC‐PCI complex before and 1 hr after PTCA. The plasma levels of D‐dimer, soluble fibrin monomer, thrombomodulin, TF and PPIC were significantly decreased 1 hr, and the plasma levels of plasmin‐α2 antiplasmin inhibitor complex 1 day after PTCA. These findings suggest that the decrease of protein C and antithrombin resulted in activation of the coagulation system. One hour after PTCA, the plasma levels of (total‐free) TF pathway inhibitor (TFPI) were significantly decreased, but the plasma levels of total and free‐TFPI were significantly increased, suggesting that comsumption of (total‐free) TFPI occurs during PTCA. Overall, these findings suggest that the hypercoagulable state improves during PTCA and that transient decrease of antithrombin, protein C, (total‐free) TFPI or plasmin‐α2 antiplasmin inhibitor complex may cause restenosis of coronary artery. Am. J. Hematol. 61:238–242, 1999.

Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site

Human blood coagulation factor X has two N-linked oligosaccharides at Asn39 and Asn49 residues and two O-linked oligosaccharides at Thr17 and Thr29 residues in the region of the factorX activationpeptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn39 and Asn49 residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri-and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Acα-2→6 residue. In addition, the structures of O-linked oligosaccharides at Thr17 and Thr29 residues were suggested to be disialylated Gal/β3GalNAc sequences by their component analyses.

Acquired Activated Protein C Resistance in Patients with Lupus Anticoagulant and Essential Thrombocythemia

The prevalence of activated protein C (APC) resistance and the antigen levels of factor V were assessed in 37 patients with lupus anticoagulant (LA), 12 with essential thrombocythemia (ET), 17 with idiopathic thrombocytopenic purpura (ITP), and in 27 cases of thrombotic complications associated with diabetes mellitus and collagen vascular disease. Blood samples taken from 30 healthy normal subjects were also available for comparison. The mean APC ratio of patients with LA (2.9 ± 1.5), ET (2.7 ± 1.2), and secondary thrombosis (2.6 ± 0.9) were significantly lower than that of the healthy control group (3.5 ± 1.0). The APC ratio of ET patients with thrombosis (2.3 ± 0.6) was significantly lower than that measured in ET cases without thrombotic complication (3.9 ± 1.9). Patients positive for LA and with thrombotic complication (1.8 ± 1.4) presented lower APC ratios than those without thrombosis (3.3 ± 1.4). Among all patients, an APC ratio lower than 2 was found in 24 cases, of which 16 had thrombotic disease, but none of them presented the factor V:R506Q mutation. The antigen levels of factor V correlated significantly with APC ratio in all patients. The results of this investigation suggest that an acquired poor anticoagulant response to APC might explain, at least in part, the thrombophilia of patients with LA and ET and that associated with diabetes mellitus or collagen disease. Key Words: APC resistance—Lupus anticoagulant—Essential thrombocythemia.