Tomio Kawasaki

Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis.

BACKGROUND Activated platelets have been recently reported to produce platelet microparticles and to enhance platelet-leukocyte interaction. The precise role of platelets in systemic inflammatory response syndrome (SIRS) has not been clarified. The objective of this study was to evaluate microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. METHODS Twenty-six patients with severe SIRS (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 12 healthy volunteers were studied. The severe SIRS was caused by trauma in 12 patients and sepsis in 14. Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding, and platelet-polymorphonuclear leukocyte (PMNL) binding were measured by flow cytometry in the presence or absence of ionomycin, N-formyl-methionyl-leucyl-phenylalanine, or anti-CD62p monoclonal antibody. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood were also measured. RESULTS Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding with or without ionomycin, and platelet-PMNL binding with ionomycin significantly increased in patients with severe SIRS in comparison with values in normal volunteers. The increased platelet-leukocyte binding in severe SIRS patients was markedly inhibited by P-selectin blockade and was not enhanced by N-formyl-methionyl-leucyl-phenylalanine. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood also increased in these patients. CONCLUSION Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. Enhanced platelet-leukocyte interaction is dependent on P-selectin expression and may be involved in the systemic inflammatory response after severe inflammatory insult.

Increased Platelet Sensitivity to Collagen in Individuals Resistant to Low-Dose Aspirin

BACKGROUND AND PURPOSE The purpose of this study was to assess individual differences in the pharmacological effects of acetylsalicylic acid (ASA) on bleeding time as measured by in vitro platelet aggregation and to examine the consistency of responses over time. METHODS We measured template IIR bleeding time and platelet aggregation in 8 healthy male volunteers before and 2 hours after ingestion of 324 mg of ASA. An individual was considered a nonresponder if his post-ASA bleeding time was not 2 SDs above his baseline bleeding time, where SD was estimated from the baseline bleeding times of the 8 volunteers. The same experiment was done after a 30-month interval. RESULTS Five volunteers were identified as ASA responders, and 3 were identified as nonresponders. Bleeding time before and after ingestion of ASA was 408+/-121 seconds (mean+/-SD) and 720+/-225 seconds, respectively, in ASA responders and 330+/-30 seconds and 330+/-52 seconds, respectively, in ASA nonresponders. The mean ED(50) for collagen-induced platelet aggregation, that is, the mean concentration of collagen that caused a response at 50% of maximum, was 0.91 microg/mL (95% CI, 0.73 to 1. 14) in ASA responders and 0.48 microg/mL (95% CI, 0.38 to 0.60) in nonresponders. When optimum concentrations of collagen, ie, concentrations that yielded 90% maximum aggregation, were used as stimuli, the mean IC(50) for ASA, that is, the mean concentration that yielded 50% inhibition, was 322.5 micromol/L (95% CI, 264.8 to 392.6) in ASA responders and 336.1 micromol/L (95% CI, 261.0 to 432. 8) in nonresponders. The variability in individual responsiveness in the second experiment remained consistent with that in the first experiment. CONCLUSIONS ASA resistance may be caused by an increased sensitivity of platelets to collagen. A platelet aggregation study specific for collagen dose response may be useful for strict selection of ASA responders for low-dose ASA therapy and for identifying ASA nonresponders for high-dose ASA therapy.

Inhibition of Experimental Abdominal Aortic Aneurysm in the Rat by Use of Decoy Oligodeoxynucleotides Suppressing Activity of Nuclear Factor κB and ets Transcription Factors

Background—Two phenomena, inflammation and matrix degradation, contribute to the progression of abdominal aortic aneurysm (AAA). Importantly, the inflammation is regulated by the transcription factor nuclear factor (NF)–&kgr;B, whereas the destruction and degradation of elastin fibers by matrix metalloproteinases (MMP) are regulated by ets. Thus, we developed a novel strategy to treat AAA by simultaneous inhibition of both NF-&kgr;B and ets by using chimeric decoy oligodeoxynucleotides (ODN). Methods and Results—AAA was induced in rats by transient aortic perfusion with elastase, whereas transfection of decoy ODN was performed by wrapping a delivery sheet containing decoy ODN around the aorta. Gel-mobility shift assay at 7 days after treatment demonstrated that both NF-&kgr;B and ets binding activity were simultaneously inhibited by chimeric decoy ODN. Transfection of chimeric decoy ODN resulted in significant inhibition of the progression of AAA such as aneurysmal dilation at 4 weeks after treatment as compared with control, accompanied by a reduction of MMP expression. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with chimeric decoy ODN. Importantly, transfection of chimeric decoy ODN demonstrated potent inhibition of aneurysmal dilatation compared with NF-&kgr;B decoy ODN alone, whereas scrambled decoy ODN had no effects. Interestingly, the migration of macrophages was significantly inhibited by chimeric decoy ODN. Conclusions—We demonstrated that inhibition of the progression of AAA was achieved by a novel strategy with chimeric decoy ODN used against NF-&kgr;B and ets in rat model. NF-&kgr;B and ets are considered to play an important role in the pathogenesis of AAA.

Activation of coagulation and fibrinolysis during surgery, analyzed by molecular markers

Activation of hemostasis during surgery was investigated in 30 elective cases, who underwent either gastric (group G) or hepatic (group H) resection by a serial determination of various molecular markers such as fibrinopeptide A (FPA), fibrinopeptide B beta 15-42 (B beta 15-42) D-dimer, thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 plasmin inhibitor complex (PIC). In both groups, the values of FPA and TAT were significantly elevated intraoperatively, indicating an occurrence of hypercoagulable state. The degree of the elevation was more marked in group H, probably due to greater tissue damage during hepatic resection. Also in both groups, the values B beta 15-42 and PIC were significantly increased during surgery, while the amount of D-dimer was within normal range in most cases, indicating the occurrence of the primary fibrinolysis. These findings are compatible with our previous observations on the postoperative changes in hemostasis. There were statistically significant but variable correlations between the values of fibrinopeptides and the enzyme-inhibitor complexes. The absolute values of the molecular markers of fibrinolysis were always higher than those of coagulation, suggesting that a considerable amount of plasmin, rather than thrombin, is released by surgical tissue damages.

Shear stress induces expression of CNP gene in human endothelial cells

To elucidate the effect of blood flow on gene transcription of C‐type natriuretic peptide (CNP), human umbilical vein endothelial cells were exposed to shear stress in a cone‐plate viscometer. Expression of CNP mRNA, evaluated by reverse transcription‐polymerase chain reaction, was markedly increased by exposure to shear stress of 24 dyne/cm2 at 3 h. The CNP mRNA level was maintained until 12 h. Thus, the present study demonstrated for the first time that shear stress induces expression of CNP gene in human endothelial cells.

Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis

INTRODUCTION Genetic deficiencies of PROS1, PROC, and SERPINC1 (antithrombin) are risk factors for deep vein thrombosis (DVT). Diagnosis of the inherited deficiencies of these three genes is sometimes difficult because of the phenotypic variability. This study was undertaken to reveal the frequency of nonsynonymous mutations of these three genes in Japanese DVT patients. PATIENTS/METHODS One hundred seventy-three DVT patients were registered by the Sub-group of Blood Coagulation Abnormality, from the Study Group of Research on Measures for Intractable Diseases. We sequenced the entire coding regions of the three genes in all DNA samples and identified the nonsynonymous mutations. RESULTS AND CONCLUSIONS For PROS1 we identified 15 nonsynonymous mutations in 28 DVT patients; for PROC, 10 nonsynonymous mutations in 17 patients; and for SERPINC1, 13 nonsynonymous mutations in 14 patients. Five patients had two mutations in PROS1 and PROC, and all of them had PROS1 K196E mutation. We previously identified one patient with a large PROS1 gene deletion. Thus, 55 out of 173 patients (32%) carried at least one genetic defect in the three genes. The PROS1 K196E mutation found in 15 Japanese DVT patients was the most prevalent. Mutations of PROC K193del and V339M were the second, each found in four patients. Our data suggested that the PROC K193del mutation caused the loss of the anticoagulant activity but not the amidolytic activity. Our effort is the first DNA resequencing study to identify the genetic variations in DVT patients without any consideration of their plasma activities and antigens. To minimize selection bias in a future evaluation of the contribution of genetic deficiency to DVT, we must recruit patients consecutively.

Hypercholesterolemia as a risk factor for deep-vein thrombosis.

Our retrospective study has shown that hyperlipidemia is a novel etiologic factor in deep-vein thrombosis (DVT) and that most of the idiopathic DVT patients were hyperlipidemic (Thrombosis Research 79, 147-151, 1995). The aim of our current study is to analyze the interrelationship between hyperlipidemia and DVT by means of a case-control study. A series of lipid parameters were analyzed using serum from 109 patients with deep vein thrombosis (DVT). One hundred nine age- and sex-matched subjects served as controls. Diagnosis of hyperlipidemia was made if the serum cholesterol level was above 220 mg/dL or if the triglyceride level was above 150 mg/dL. Among several types of hyperlipidemia examined, the risk factor associated with the highest estimated odds ratio was carriage of hypercholesterolemia associated with hypertriglyceridemia (odds ratio 5.1) followed in order by hypercholesterolemia without hypertriglyceridemia (odds ratio 2.6) and hypertriglyceridemia without hypercholesterolemia (odds ratio 0.9). These findings support the hypothesis that hypercholesterolemia plays an important role in the pathogenesis of DVT.

The effects of calpeptin (a calpain specific inhibitor) on agonist induced microparticle formation from the platelet plasma membrane.

Platelets activated by various agonists produce formation of vesicles shed from the plasma membrane (microparticles). However, the mechanism of microparticle (MP) formation has not been clarified yet. The aim of the present study was to determine the possibility of involvement of calpain (a Ca(2+)-dependent thiol protease) in MP formation. Washed platelets preincubated with calpeptin, a cell permeable calpain specific inhibitor, or with a vehicle were activated by thrombin plus collagen or by calcium ionophore A23187. Flow cytometry was used to detect the amount of microparticle formation by using murine monoclonal antibodies against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate labeled goat anti-mouse IgG. MP formation stimulated either by thrombin plus collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A23187 reached a plateau in approximately 5 min after activation, whereas that from platelets activated by thrombin plus collagen reached a plateau at 30 min following the stimulation. These time sequences corresponded well with those of degradation of actin-binding protein (ABP), a well known substrate of calpain, of platelets activated by these two stimulations. However, the inhibition of MP formation by calpeptin was more marked in the early stage (within 10 min) than in the late stage (after 30 min) of platelet activation. At 30 min after platelet activation by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 microM calpeptin, which inhibited hydrolysis of ABP almost completely. Our data suggest the involvement of calpain in the early stage (especially within 10 min) of microparticle formation.

Regression of Abdominal Aortic Aneurysms by Simultaneous Inhibition of Nuclear Factor κB and Ets in a Rabbit Model

Because current therapy to treat abdominal aortic aneurysm (AAA), and particularly to manage small AAA, is limited to elective surgical repair, we explored less invasive molecular therapy by simultaneous inhibition of the transcription factors nuclear factor (NF)&kgr;B and ets using a decoy strategy. Both NF&kgr;B and ets were shown to be markedly activated in human AAA. In addition, NF&kgr;B- and ets-positive cells were increased in the aneurysm wall, and a part of the expression of NF&kgr;B and ets was detected in migrating macrophages. Thus, we used chimeric decoy oligodeoxynucleotides (ODNs) containing consensus sequences of both NF&kgr;B and ets binding sites to treat AAA. Inhibitory effects of chimeric decoy ODNs on matrix metalloproteinase-1 and -9 expression were confirmed by ex vivo experiments using a human aorta organ culture. To examine the regressive effect in a rabbit already-formed AAA model, transfection by wrapping a delivery sheet containing chimeric decoy ODNs around the aneurysm was performed 1 week after incubation with elastase. Importantly, treatment with chimeric decoy ODNs significantly decreased the size of AAA. Interestingly, significant preservation of elastic fibers was observed with chimeric decoy ODN treatment, accompanied by a reduction of matrix metalloproteinase-2 and -9 and induction of macrophage apoptosis. Regression of AAA was also associated with an increase in elastin and collagen type I and III synthesis in the aneurysm wall. Minimally invasive molecular therapy targeted to the inhibition of NF&kgr;B and ets is expected to be useful for AAA through the rebalance of matrix synthesis and degradation.

Frequency of natural coagulation inhibitor (antithrombin III, protein C and protein S) deficiencies in Japanese patients with spontaneous deep vein thrombosis.

One hundred and thirteen consecutive Japanese patients with deep venous thrombosis (DVT) were studied for the incidences of antithrombin III (AT-III), protein C (PC) and protein S (PS) deficiencies, and the results were compared with those of normal subjects. Ten of the 392 normal Japanese subjects were found with PS deficiency (n= 8, 2.02%) or PC deficiency (n= 2, 0.5%). PS deficiencies comprised type I (1/8, 12.5%), type II (4/8, 50%), and type III (3/8, 37.5%). All PC deficiencies were type I. Among patients with DVT, 32 (28.3%) were deficient in AT-III, PC and PS. These patients consisted of two AT-III deficiency (1.77%), nine PC deficiency (7.96%), 20 PS deficiency (17.7%), and one combined deficiency of PC and PS (0.88%). Both of the patients with AT-III deficiency were classified as type II, all those with PC deficiency as type I, and those with PS deficiency as type I in 25% (5/20), type II in 55% (11/20) and type III in 20% (4/20). The frequency of PC and PS deficiencies in patients with DVT were 15.6 and 7.38 times the control population frequency, respectively, and this difference was statistically significant (P< 0.05). These data suggest that the Japanese population has a high frequency of PC and PS deficiencies. We recommend that PS activity should be measured for screening of thrombosis since type II deficiency accounted for approximately 50% of PS deficiency cases in both patients and the normal group in the Japanese.