Effects of sarpogrelate hydrochloride on adenosine diphosphate- or collagen-induced platelet responses in arteriosclerosis obliterans.

To evaluate the effects of the 5-HT2 receptor antagonist sarpogrelate hydrochloride (sarpogrelate) on platelet responses in arteriosclerosis obliterans (ASO), we examined platelet aggregation and its relationships to platelet-derived growth factor (PDGF), soluble P-selectin (sP-selectin), and transforming growth factor-beta 1 (TGF-beta1). Circulating plasma levels of PDGF and sP-selectin in 13 patients with ASO after 1 week of medication with sarpogrelate were significantly lower than those before medication. In contrast, circulating plasma levels of TGF-beta1 after medication were significantly higher than those before medication. When platelet-rich plasma obtained from ASO patients after medication was stimulated with adenosine diphosphate (ADP) or collagen, platelet aggregation was suppressed compared with rates before medication. Significant decreases in levels of PDGF, sP-selectin and TGF-beta1 released from platelets in response to 5 micromol/l ADP and 1 microg/ml collagen after taking of sarpogrelate were found. There were close correlations between platelet aggregation and respective molecules released from platelets. In conclusion, since platelet activation is involved in pathogenesis of thrombotic disease, sarpogrelate may suppress the development of obstructive arteriosclerosis. PDGF and TGF-beta1, as well as sP-selectin, appear to be useful markers for clinical evaluation of anti-platelet drugs.

Rapid detection of the 22q11.2 deletion with quantitative real-time PCR.

We report the detection of 22q11.2 deletions using TaqMan(TM)PCR-based gene dosage analysis of patients previously diagnosed by fluorescent in situ hybridization (FISH). We performed quantitative PCR of the UFD1L gene from this region and showed a 99.7% statistical correlation between the two methods. The advantages of this method over FISH include: (i) rapidity; (ii) ease; (iii) relatively low cost and; (iv) the small quantities of DNA needed.

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol.

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the alpha-granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 microM). Platelet-rich plasma was stimulated by ADP (1 and 5 microM) or collagen (5 microg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 microM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 microM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 microM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 microM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.

Influence of preoperative nutritional state on inflammatory response after surgery.

To investigate whether the preoperative nutritional state influences the postoperative inflammatory reaction and immunity, we grouped patients whose postoperative nutritional support was performed by total parenteral nutrition into the good nutritional state group (group I) and the latent protein-calorie malnutrition suggested group (group II) based on the preoperative rapid turnover protein (RTP). Nutritional markers markedly decreased after surgery and recovered almost to preoperative levels on postoperative day (POD-) 7 in groups I and II. Nutritional markers on POD-7 in group II were significantly lower than those in group I (RTP, P < 0.001; albumin, P < 0.05). After surgery, levels of interleukin-6 (IL-6), C-reactive protein (CRP), and polymorphonuclear (PMN-) elastase were higher in group II than in group I (P < 0.01). In groups I and II, IL-6 and interleukin-8 (IL-8) rose before the remarkable elevation of CRP and PMN-elastase. In group I, all the nutritional markers showed a negative correlation with CRP and PMN-elastase. Further, a positive correlation was observed between IL-6 and CRP and between IL-8 and PMN-elastase. In conclusion, evaluation of the preoperative nutritional state appears to be very important for the prediction of postoperative complication.

Inhibitory effects of H2-receptor antagonists on platelet function in vitro.

To evaluate in vitro inhibitory effects of four types of histamine H2-receptor antagonist (H2-receptor antagonists), famotidine, roxatidine, cimetidine and ranitidine, on platelet function, we examined aggregating potency and P-selectin levels with agonist-induced aggregation. Ranitidine and cimetidine inhibited, in concentration of 0.35 mM, the secondary aggregation induced by 5 microM adenosine diphosphate (ADP), the aggregation induced by 1 microg/mL collagen and 3 microM arachidonic acid. All of H2-receptor antagonists inhibited, in concentration of 1.4 mM, the aggregation induced by ADP, collagen and arachidonic acid. Ranitidine and cimetidine reduced markedly, in same concentration, P-selectin levels after induction of aggregation by 5 microm ADP, 1 microg/mL collagen and 3 microM arachidonic acid. When classified by the strength of inhibitory action, ranitidine and cimetidine were strong, followed by famotidine and roxatidine. It is considered that inhibitory effects of H2-receptor antagonists on platelet function are weaker than those of acetylsalicylic acid (ASA), since ASA inhibited platelet aggregation in concentration of 100 microM. No relationship was observed between inhibitory effects of H2-receptor antagonists on platelet aggregation induced by above agonists and the presence or absence of imidazole ring in the chemical structure.

Effects of sarpogrelate hydrochloride on platelet aggregation, and its relation to the release of serotonin and P-selectin.

Inhibitory effects of sarpogrelate hydrochloride (sarpogrelate), a 5-HT2 receptor antagonist, on platelet aggregation was examined as well as the relationship to serotonin and P-selectin, a platelet alpha-granule membrane glycoprotein. Platelet aggregation was induced by simultaneous addition of collagen (0.06-0.12 microg/ml), which did not induce aggregation alone, and serotonin (0.88 micromol/1) to platelet-rich plasma (PRP). The PRP was obtained from healthy volunteers and percentage maximum aggregation (MA) was measured. Serotonin levels and P-selectin levels in the supernatant of PRP after aggregation were determined. When vehicle-treated PRP was stimulated in the aforementioned manner, platelet aggregation dependent on collagen concentration was induced. Serotonin levels and P-selectin levels were also dependent on collagen concentration. Sarpogrelate (10(-6) to 10(-4) mol/l) inhibited such aggregation dose-dependently, and decreased serotonin levels and P-selectin levels in a dose-dependent manner. There were close correlations between MA and serotonin levels, MA and P-selectin levels, as well as serotonin and P-selectin levels. These results suggest that extracellular release of serotonin and P-selectin from platelets was caused by induction of aggregation, and these responses were suppressed by sarpogrelate.

Relationship between changes in F1+2 and TAT levels and blood coagulation early after prosthetic valve replacement.

Prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin III complex (TAT), D-dimer and other coagulation-related factors were measured over time in 48 patients who underwent prosthetic valve replacement and subsequent anticoagulant therapy, in order to evaluate the dynamics of thrombin generation. Before surgery, levels of both F1 + 2 and TAT were high, indicating enhanced thrombin generation. On the 7th day after initiation of postoperative administration of warfarin, F1 + 2 was significantly lower than preoperatively, while TAT tended to be increased. Even on the 21st day, levels of both F1 + 2 and TAT were still high. Despite the administration of warfarin, thrombin generation increased in these patients. After surgery, D-dimer level was increased, indicating hyperfibrinolysis. On the 21st day after initiation of warfarin administration, abnormally high levels of F1 + 2 were observed in 4 of the 48 patients. In these 4 patients, thrombin generation was markedly increased, suggesting that they were in a preliminary stage of thrombogenesis. Antiplatelet therapy did not affect the levels of F1 + 2 or TAT. Our findings suggest that both F1 + 2 and TAT levels are useful for evaluation of enhanced coagulability. D-dimer is also considered to be useful as a parameter of fibrinolysis. F1 + 2 is less affected by surgical invasion than TAT, and therefore appeared to reflect coagulability more accurately.

Inhibitory effects of antibiotics on platelet aggregation in vitro.

1. We evaluated in vitro inhibitory effects of six types of antibiotic, aztreonam (AZT), cefamandole (CMD), cefmetazole (CMZ), cefotiam (CTM), flomoxef (FMOX) and latamoxef (LMOX), on platelet aggregation, using healthy volunteers' blood. Four types--FMOX, LMOX, CTM and CMD--inhibited, in concentration of 2500 micrograms/ml, the secondary aggregation induced by 3.0 microM adenosine diphosphate (ADP), and also inhibited the aggregation induced by 0.5 micrograms/mi collagen. AZT in the same concentration, did not inhibit the aggregation induced by collagen, and it inhibited only ADP-induced aggregation. CMZ, in the same concentration, inhibited neither of the two aggregations. 2. The inhibitory effects of the antibiotics on collagen-induced aggregation were dependent on the concentration of respective antibiotics. When classified by the strength of inhibitory action, LMOX and FMOX were strong, followed by CTM and CMD. The action of AZT and CMZ was weak. In particular, LMOX showed a 32% inhibitory effect at concentration of 50 micrograms/ml, a level near the blood concentration obtained with clinical usual dose. 3. No relationship was observed between inhibitory effects of antibiotics on ADP- or collagen-induced aggregation and the presence or absence of carboxyl group and/or N-methyltetrazolethiol group in the chemical structure.

Prophylactic action of allopurinol against chemotherapy-induced stomatitis--inhibition of superoxide dismutase and proteases.

The activities of superoxide dismutase (SOD) and several proteases were measured in kidney of mice treated with allopurinol in order to elucidate the mechanism of prophylactic action of allopurinol against chemotherapy-induced stomatitis. The following results were obtained. Following 3 day administration of allopurinol 20 mg/day per os (Group C), the concentrations of allopurinol and oxipurinol in the renal tissue were 203.9 +/- 52.1 and 1141.7 +/- 194.8 micrograms/g, respectively. The SOD activity was significantly lower in Group C than in the untreated control group (p < 0.01). The enzyme activities of papain and trypsin were suppressed in Group C. However, the other proteases tested were not affected by the administration of allopurinol, indicating only weak anti-protease action of allopurinol. These results suggest that allopurinol may be effective to prevent chemotherapy-associated stomatitis via both direct and indirect actions to oral mucosa, that include inhibitory actions on xanthine oxidase as well as protease.

[Significance of combined use of anticoagulants and antiplatelet agents in the early stage after prosthetic valve replacement].

Patients who had undergone prosthetic valve replacement were treated with warfarin (anticoagulant) alone or in combination of ticlopidine (200 mg/day) or aspirin (81 mg/day) (anti-platelet agents). The study of blood coagulation factors and platelet aggregation were carried out with these cases. 1) The patients (n = 24) receiving warfarin for 21 days after prosthetic valve replacement revealed marked increases in PIVKA-II and vitamin K1-epoxide. The protein C activity was significantly lower than that before the operation. High levels of more than 5 ng/ml of TAT were found before operation and after warfarin administration for 21 days. 2) Warfarin did not affect platelet aggregation, whereas ticlopidine inhibited ADP-induced platelet aggregation and aspirin inhibited both collagen-induced and arachidonic acid-induced aggregation. In conclusion, combined use of anticoagulants and antiplatelet agents after prosthetic valve replacement will suppress not only the blood coagulation but also the platelet aggregation systems.

Anticoagulant effects of warfarin and kinetics of K vitamins in blood and feces.

Patients (40 cases) were treated with daily dosage of warfarin of 2-7 mg after being undergone artificial valve replacements. Twenty one days after administration of warfarin, we examined the patients for kinetics of K vitamins and vitamin K-dependent coagulation factors in blood, and intestinal flora in feces, as well as the relationship between K vitamins and coagulation activity. The following results were obtained. (1) In warfarin-administered patients (Group B), blood levels of vitamin K1 and menaquinone-7, a vitamin K2 homologue, were similar to those in non-warfarin-administered patients. Therefore, administration of warfarin did not significantly decreased the levels. (2) In patients selected randomly from Group B (Group C), the vitamin K1 level in feces was higher than that in non-warfarin-administered patients. The menaquinone-7 level in feces was similar to that in non-warfarin-administered patients. For the total counts of bacteria and the detection rate of vitamin K2-producing bacteria, there was no significant difference between Group C and non-warfarin-administered patients. (3) The above mentioned results of (1) and (2) suggest that it is important for development of anticoagulant effects by warfarin to inhibit conversion from vitamin K1 to reduced vitamin K1, as well as to inhibit the reducing process from vitamin K1-epoxide to vitamin K1. (4) Vitamin K1-epoxide, a metabolite of vitamin K1, appeared in blood after administration of warfarin; there was a lower correlation between the blood level of vitamin K1-epoxide and the warfarin dosage. Further, PIVKA-II appeared in blood after administration of warfarin; there was a inverse lower correlation between the level of PIVKA-II and HPT, and between PIVIKA-II and TT. In conclusion, it has been clarified that vitamin K1-epoxide and PIVKA-II are useful parameters to evaluate anticoagulant effect of warfarin.

Variations in warfarin concentration in blood and coagulant factors after artificial valve replacement.

A proper dosage of warfarin after artificial cardiac valve replacements has been determined as an indication of coagulation activity. In some cases, the coagulation activity cannot be maintained within the therapeutic range because Prothrombin time (PT) values deviated from Thrombotest (TT) values. Here we studied each relationship between warfarin concentrations in blood, coagulation activity, vitamin Ks and other coagulation-related agents which were measured in patients administered warfarin after artificial cardiac valve replacements and whose coagulation activity was maintained within a therapeutic range, and in patients whose PT values deviated from TT values. The obtained results were interpreted as follows. 1) There was a high correlation between values from Thromborel S (a clot method) and values from Chromoquick (a method using a synthesized substrate). Since TT values had little variations compared to PT values, the former was an excellent indication of coagulation activity. 2) There was no correlation between warfarin concentrations in blood and coagulation activity in the group in which warfarin concentrations in blood vary within a day (Group B); but there was highly reverse correlation in the group in which warfarin concentrations in blood vary with days (Group A). 3) There was good correlation between warfarin concentrations and PIVKA-II concentrations in blood in Group A. Further, the concentration of Gla-Protein C was one microgram/ml or less on the fourth day from the initiation of warfarin administration. 4) For example, a patient whose PT value of 67% deviated from TT value of 19% on the eleventh day from the initiation of warfarin administration had vitamin K1-epoxide concentration of 2.68 ng/ml and MK-7 concentration of 1.26 ng/ml on the same day, respectively, which were higher levels than those in Group A.