OBJECTIVE: To investigate the effect of protein kinase A (PKA) activation on aggregation funetion of platelets in vitro. METHODS: The peripheral blood of healthy adults were collected, and the washed platelets were gained from collected peripheral blood. The washed platelets were treated with PKA activator Forskolin, then the platelet aggregation was induced by using Ristocetin, Thrombin, Collagen and ADP respectively, the platelet aggregation level was detected by the platelet aggregator. RESULTS: Compared with the controls, 5 μmol/L forskolin significantly inhibited ADP and collagen-induced platelet aggregation (P＜0.001), and showed mild inhibiting effect on Thrombin-induced platelet aggregation (P＜0.05). 2.5-10 μmol/L forskolin significantly inhibited ADP and Collagen -induced platelet aggregation (P＜0.001); but not showed significantly inhibitory effects on Ristocetin-induced platelet aggregation (P＞0.05). CONCLUSION PKA activation inhibits agonists-induced platelet aggregation.
Blood Platelets ; Cyclic AMP-Dependent Protein Kinases ; Humans ; Platelet Aggregation ; Platelet Aggregation Inhibitors ; Ristocetin ; Thrombin

BACKGROUND: Although apoptosis occurs in nucleated cells, studies show that this event also occurs in some anucleated cells such as platelets. During storage of platelets, the viability of platelets decreased, storage lesions were observed, and cells underwent apoptosis. We investigated the effects of caspase-3 inhibitor on the survival and function of platelets after different periods of storage. METHODS: Platelet concentrates were obtained from the Iranian Blood Transfusion Organization in plastic blood bags. Caspase-3 inhibitor (Z-DEVD-FMK) was added to the bags. These bags along with control bags to which no inhibitor was added were stored in a shaking incubator at 22degrees C for 7 days. The effects of Z-DEVD-FMK on the functionality of platelets were analyzed by assessing their ability to bind to von Willebrand factor (vWF) and to aggregate in the presence of arachidonic acid and ristocetin. Cell survival was surveyed by MTT assay. RESULTS: At day 4 of storage, ristocetin-induced platelet aggregation was significantly higher in the inhibitor-treated (test) than in control samples; the difference was not significant at day 7. There was no significant difference in arachidonic acid-induced platelet aggregation between test and control samples. However, at day 7 of storage, the binding of platelets to vWF was significantly higher in test than in control samples. The MTT assay revealed significantly higher viability in test than in control samples at both days of study. CONCLUSION: Treatment of platelets with caspase-3 inhibitor could increase their functionality and survival.
Apoptosis ; Arachidonic Acid ; Blood Platelets* ; Blood Transfusion ; Caspase 3* ; Cell Survival ; Incubators ; Plastics ; Platelet Aggregation ; Ristocetin ; von Willebrand Factor

BACKGROUND AND OBJECTIVES: Cilostazol, a selective inhibitor of phosphodiesterase III (PDE III), prevents inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP) and irreversibly inhibits platelet aggregation and vasodilation. Hence, we performed this prospective randomized study to evaluate the clinical effects of additional cilostazol administration in patients receiving dual antiplatelet therapy after drug-eluting stent (DES) insertion. SUBJECTS AND METHODS: Between December 2003 and June 2006, we enrolled a total 603 consecutive patients who underwent successful percutaneous coronary intervention (PCI) with DES insertion at Dong-A University Hospital. Study patients received dual antiplatelet therapy (aspirin and clopidogrel, n=301) for at least six months or dual antiplatelet therapy (six months) combined with cilostazol medication for one month (triple therapy, n=302) after PCI. We investigated the incidence of major adverse cardiac events (MACE) at one month and six months after the initiation of medical therapy. MACE was defined as a composite of death, myocardial infarction (MI), stent thrombosis, and target lesion revascularization (TLR). Platelet function was evaluated in 66 patients (dual therapy group, n=40; triple therapy group, n=26) using a Chrono-Log platelet aggregometer and the VerifyNow P2Y12 assay system. RESULTS: The MACE rate was 0.66% in the triple therapy group (death only, 0.67%) and 1.67% in the dual therapy group (death, 0.67%; MI, 0.67%; stent thrombosis, 0.99%; TLR, 0.99%) at one month after PCI (p=0.087). At six months, there were no differences in the MACE rate between the two groups (triple group vs. dual group=2.65% vs. 3.99%, p=0.864). In laboratory tests, platelet aggregation induced by agonists of ADP (27.92+/-13.04% vs. 40.9+/-15.78%, p=0.0008), collagen (13.73+/-6.95% vs. 27.43+/-14.87%, p=0.03), and epinephrine (10.38+/-7.82% vs. 15.5+/-10.45%, p=0.0000) were lower in the triple therapy group versus the dual therapy group. However, platelet aggregation induced by agonists of arachidonic acid (3.23+/-1.07% vs. 3.78+/-2.12%, p=0.23) and ristocetin (29.19+/-35.55% vs. 44.78+/-32.65%, p=0.07) and aspirin reaction unit (412.96+/-96.25 vs. 427.93+/-76.24, p=0.48) measured by VerifyNow were not different in the triple group versus the dual group. CONCLUSION: Additional administration of cilostazol did not decrease the MACE rate when compared to dual therapy six months after PCI in patients with DES.
Adenosine Diphosphate ; Adenosine Monophosphate ; Arachidonic Acid ; Aspirin ; Blood Platelets ; Collagen ; Cyclic Nucleotide Phosphodiesterases, Type 3 ; Drug-Eluting Stents ; Epinephrine ; Humans ; Incidence ; Myocardial Infarction ; Percutaneous Coronary Intervention ; Platelet Aggregation ; Prospective Studies ; Ristocetin ; Second Messenger Systems ; Stents ; Tetrazoles ; Thrombosis ; Ticlopidine ; Vasodilation

BACKGROUND: The limit and the optimal method of the cryopreservation of platelets have not been determined. Moreover, the functional changes platelets after cryopreservation were not clearly defined. This study was conducted to determine the limit and optimal method for cryopreservation of platelet concentrates. METHODS: We compared the recovery, expression of membrane GpIb, GpIIb/IIIa, and aggregatory function of the platelets preserved in three different conditions. Platelet samples were collected from four healthy volunteer donors by apheresis, and placed in 22degrees C agitator for standard preservation. For cryopreservation, after treating 5% DMSO, platelets were either inserted directly in -80degrees C freezer or in liquid nitrogen after computer-controlled rate freezing. After storage for 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, and 12 weeks, platelets were thawed and analyzed for the evaluation of in vitro functions. RESULTS: Platelets preserved at 22degrees C or cryopreserved with each condition displayed equivalent recovery (90%). With each cryopreservation procedures, platelets showed moderate loss of GpIb and retained more than 90% of GpIIb/IIIa in comparison with fresh platelets. At the third week, loss of GpIb in the directly frozen platelets was augmented compared with those of controlled rate frozen group. The aggregatory response to ristocetin began to decrease drastically after storage for 5 days in platelets frozen by each procedures and to less than 5% at 12 weeks of storage. However, controlled rate frozen platelets retained more aggregatory response to ristocetin and surface GpIb expression than those of directly frozen platelets at 3, 4, 12 weeks of storage. CONCLUSION: This study showed the possibility of moderate preservation of in vitro functions of frozen-thawed platelets after 12 weeks of storage compared with those of the liquid stored 5-day old platelets.
Blood Component Removal ; Blood Platelets ; Cryopreservation* ; Dihydroergotamine ; Dimethyl Sulfoxide ; Freezing ; Healthy Volunteers ; Humans ; Membrane Glycoproteins ; Membranes ; Nitrogen ; Ristocetin ; Tissue Donors

PURPOSE: von Willebrand disease is a common inherited bleeding disorder characterized by high degree of variable clinical presentation due to either quantitative or qualitative defects in von Willebrand factor. Its incidence in Korea is not well studied while that in western countries is extensively studied. METHODS: We classified 16 cases of vWD from 14 unrelated families based on vWF antigen, ristocetin cofactor activity, factor VIII activity and vWF multimeric patterns analysed by agarose gel electrophoresis, according to a revised classification by ISTH. RESULTS: There were 12 cases (75%) of type 1 vWD or 2M/2N with normal multimeric pattern, 3 cases (18.75%) of type 2 vWD lacking high molecular weight multimers and only 1 case of type 3 vWD with no multimers. CONCLUSION: The proportion of each vWD subtype in Korea is similar to that in western countries, however, accurate diagnosis based on ristocetin induced platelet aggregation test, factor VIII binding assay and molecular genetic diagnosis seems to be necessary for a more complete classification of vWD.
Classification ; Diagnosis ; Electrophoresis, Agar Gel ; Factor VIII ; Hemorrhage ; Humans ; Incidence ; Korea* ; Molecular Biology ; Molecular Weight ; Platelet Aggregation ; Ristocetin ; von Willebrand Disease, Type 3 ; von Willebrand Diseases* ; von Willebrand Factor

Glanzmann's thrombasthenia is a rare autosomal recessive hemorrhagic disorder of platelet function with missing or abnormal platelet plasma membrane glycoprotein IIb-IIIa, which functions as a receptor for fibrinogen. We have experienced a case of thrombasthenia in a 6-year-old female whose chief complaints were easy bruising, frequent epistaxis, arthralgia and swelling of the right ankle joint. Bleeding time was prolonged in the presence of normal platelet levels and the platelet aggregation test showed lack of aggregation after exposure to ADP, epinephrine and collagen, but showed an aggregation response to ristocetin. Platelet analysis by flow cytometry is a successful alternative rapid diagnostic technique for Glanzmann's thrombasthenia patients as well as for carriers of this disease. Flow cytometry technique provides an effective tool for investigating platelet function defects caused by altered expression or deficiency of platelet surface proteins.
Adenosine Diphosphate ; Ankle Joint ; Arthralgia ; Bleeding Time ; Blood Platelets ; Cell Membrane ; Child ; Collagen ; Epinephrine ; Epistaxis ; Female ; Fibrinogen ; Flow Cytometry* ; Glycoproteins ; Hemorrhagic Disorders ; Humans ; Membrane Proteins ; Platelet Aggregation ; Ristocetin ; Thrombasthenia*

Glanzmann's thrombasthenia is a rare autosomal recessive hemorrhagic disorder of platelet function with missing or abnormal platelet plasma membrane glycoprotein IIb-IIIa, which functions as a receptor for fibrinogen. We have experienced a case of thrombasthenia in a 6-year-old female whose chief complaints were easy bruising, frequent epistaxis, arthralgia and swelling of the right ankle joint. Bleeding time was prolonged in the presence of normal platelet levels and the platelet aggregation test showed lack of aggregation after exposure to ADP, epinephrine and collagen, but showed an aggregation response to ristocetin. Platelet analysis by flow cytometry is a successful alternative rapid diagnostic technique for Glanzmann's thrombasthenia patients as well as for carriers of this disease. Flow cytometry technique provides an effective tool for investigating platelet function defects caused by altered expression or deficiency of platelet surface proteins.
Adenosine Diphosphate ; Ankle Joint ; Arthralgia ; Bleeding Time ; Blood Platelets ; Cell Membrane ; Child ; Collagen ; Epinephrine ; Epistaxis ; Female ; Fibrinogen ; Flow Cytometry* ; Glycoproteins ; Hemorrhagic Disorders ; Humans ; Membrane Proteins ; Platelet Aggregation ; Ristocetin ; Thrombasthenia*

PURPOSE: Aspirin(acetylsalicylic acid) has been used to treat unstable angina and acute myocardial infarction in adults and Kawasaki disease in children. The antithrombotic effect of aspirin was attributed to its ability to inhibit platelet aggregation by inhibiting platelet cyclooxygenase, which leads to decreased thromboxane synthesis. The purpose of this study was to evaluate the effect on the platelet aggregation by low dose aspirin in Kawasaki patients and to learn the side effects of low-dose aspirin. METHODS: Fifty patients with Kawasaki disease who were treated with low-dose aspirin, and 22 normal children were studied from Jan. 1996 to Dec. 1997. The platelet count, bleeding time, clotting time, platelet aggregation test(induced by ADP, epinephrine, collagen and ristocetin) and blood aspirin level by colorimetric method were checked. RESULTS: The platelet count, bleeding time, and clotting time in the patient group were not significantly different from the control group. The mean maximum platelet aggregation was 54.4+/-12.8% induced by ADP, 15.9+/-11.7% by epinephrine, 55.5+/-23.8% by collagen, 52.6+/-32.2% by ristocetin in the patient group. It was significantly lower than the control group(P<0.05). The mean blood aspirin level in the patient group was 5.4+/-3.7mg/dl. Side effects of low-dose aspirin were bruise, epistaxis and hematuria. CONCLUSION: Low-dose aspirin therapy in patients with Kawasaki disease inhibited platelet aggregation, but attention would be needed because of the tendency to bleed in these patients. Further investigations should be focused on the subject such as the onset of the maximal antiplatelet effect and time needed for the recovery of platelet function.
Adenosine Diphosphate ; Adult ; Angina, Unstable ; Aspirin* ; Bleeding Time ; Blood Platelets* ; Child ; Collagen ; Contusions ; Epinephrine ; Epistaxis ; Hematuria ; Humans ; Mucocutaneous Lymph Node Syndrome* ; Myocardial Infarction ; Platelet Aggregation* ; Platelet Count ; Prostaglandin-Endoperoxide Synthases ; Ristocetin

BACKGROUND: The purpose of this study is to investigate the prevalence of impaired platelet responsiveness to epinephrine in healthy subjects. Also, we compared the platelet aggregability in response to various agonists in normal population. METHODS: A total of 156 healthy subjects aged 21 to 57 years were investigated for the evidence of impaired responsiveness to epinephrine. Aggregometer PACKS-4 (Platelet Aggregation Chromogenic Kinetic System-4, Helena, Beaumont, USA) was used for platelet function test. Aggregating agonists (Helena Haemostasis Systems, UK) used in the study were consisted of ADP (10 micrometer), collagen (10 microgram/mL), epinephrine (300 micrometer) and ristocetin (1500 microgram/mL). Population showing platelet aggregability with more than 60% activity was classified as normal group, while aggregability with less than 20% as impaired responsiveness. RESULTS: Of 156 healthy subjects, 20.5% (32/156) showed impaired responsiveness, while 33.9% (53/156) revealed decreased aggregability with the activity of less than 60% to epinephrine. The mean of maximal percent aggregating activity for collagen was 90.5+/-11.4% and that of epinephrine was 66.5+/-34.4%. The mean aggregation activity (84.4+/-11.8%) for ADP in subjects showing normal response to epinephrine was significantly higher, compared with that (65.7+/-16.2%) of impaired responsiveness group to epinephrine (P<0.01). CONCLUSION: Impaired responsiveness to epinephrine, which is observed in healthy subjects, appears to be a kind of normal variant reaction. And this abnormality is not considered to be associated with any evident bleeding disorders.
Adenosine Diphosphate ; Blood Platelets* ; Collagen ; Epinephrine* ; Hemorrhage ; Platelet Aggregation ; Platelet Function Tests ; Prevalence* ; Ristocetin

BACKGROUND: Essential thrombocythemia (ET) is a rare chronic myeloproliferative disorder characterized by an extremely high platelet count in the circulating blood and abnormal proliferation of the megakaryocytes in bone marrow, resulting in splenomegaly, thromboembolic or hemorrhagic complications. We studied the presence of nuclear hyperploidy of the megakaryocytes in bone marrow, the presence of abnormal response to the individual reagent on platelet aggregation test, and its clinical implication. METHODS: We analyzed the 43 cases of ET at the Asan Medical Center between January, 1989 and March, 1999. The Polycythemia Vera Study Group criteria were used to diagnose ET. RESULTS: Nuclear hyperploidy was observed at 43 cases (100%). Platelet aggregation test was done at 32 (74.4%) cases, of which 27 (84.4%) cases showed abnormal response to more than one reagent, 16 (50%) cases to more than two reagents. Abnormal response to epinephrine and collagen was most common, but 5 cases showed normal response. By individual reagent, 1 (3%) cases to adenosine diphosphate, 1 (3%) case to ristocetin, 22 (69%) cases to epinephrine, 19 (59%) cases to collagen showed abnormal response. CONCLUSION: We observe that nuclear hyperploidy of the megakaryocyts and abnormal response on platelet aggregation test are frequent in ET in this study.
Adenosine Diphosphate ; Blood Platelets* ; Bone Marrow ; Chungcheongnam-do ; Collagen ; Epinephrine ; Indicators and Reagents ; Megakaryocytes* ; Myeloproliferative Disorders ; Platelet Aggregation* ; Platelet Count ; Polycythemia Vera ; Ristocetin ; Splenomegaly ; Thrombocythemia, Essential*