Collagen ADP Research Articles

Primary hemostasis in patients treated with LDL-apheresis for severe familiar hypercholesterolemia: A prospective pilot trial using PFA-100 analysis to rationalize therapeutic LDL-apheresis procedure

LDL-apheresis is a method of extracorporeal elimination of serum LDL-cholesterol used for treating patients with severe hyperlipidemia resistant to diet and pharmacotherapy. A practically applicable marker that may possibly be used to ascertain the efficacy of this treatment in lowering the activity of atherosclerosis are still to be found and remains an unresolved problem. Activity of primary hemostasis plays an important role in the process of developing atherosclerotic complications. This fact led us to hypothesize that the investigation of primary hemostatic activity might be a useful marker for monitoring LDL-apheresis efficacy. The aim of this work was to verify this hypothesis.Methods and patients: Commercial analyzer Dade Behring PFA-100, Germany (PFA, platelet function analysis) was used for all investigations. This analyzer enables quantitative measurement of platelet-mediated hemostasis in uncoagulated (citrated) blood. The method simulates platelet activation by mechanical stress (shear stress), and also simulates contact of platelets with collagen. A total of nine long-term treated patients with familial hypercholesterolemia were included in the study group (4 females and 5 males). Ages ranged from 17 to 59 years (average 46.4, median 55). Two patients had homozygous hypercholesterolemia. Eighteen sample pairs were examined using collagen/epinephrine (COL/EPI) membrane and 17 pairs were examined using collagen/ADP (COL/ADP) membrane, the total number of samples amounted to 70.Results: Closure time (CT) values were prolonged after separation in all cases but CT prolongation was not statistically significant (p < 0.14). No differences between homozygous and heterozygous patients were found (p < 0.05).Conclusion: Investigation of primary hemostasis using PFA-100 analyzer is not a suitable marker and should not be used to determine the optimal intensity of individual LDL-apheresis procedures.

Abstract 2214: The Aptamer ARC1779 Is A Potent And Specific Inhibitor Of von Willebrand Factor (vWF) Mediated Ex Vivo Platelet Function In ST-elevation (STEMI) and non-ST elevation (NSTEMI) Acute Myocardial Infarction (AMI)

Background: ARC1779 is an aptamer which blocks the A1 domain binding of the vWF A1 domain to platelet GPIb receptors that is now in development for the treatment of AMI. vWF is increased in the elderly and in the setting of AMI, as reflected in higher vWF levels in circulation and in increased shear-dependent platelet function as measured by the platelet function analyzer (PFA-100) and cone and plate analyzer (IMPACT). Conventional therapy of AMI partially reduces platelet activation and aggregation, but does not address excessive vWF activity or platelet adhesion. Methods: We studied the ex vivo dose response curves for ARC1779 on PFA-100 and IMPACT platelet function tests, agonist-induced platelet aggregation, and vWF activity (free A1 domain sites) of patients with AMI on standard treatment including aspirin and clopidogrel (n=40), young (n=20) and elderly controls (n=20). Results: ARC1779 fully blocked collagen ADP induced platelet plug formation as measured by PFA-100 with an IC100 of ~ 1–2 mcg/mL with citrate anticoagulation, and 3–5 mcg/mL with hirudin anticoagulation. ARC1779 fully blocked shear-dependent platelet adhesion measured by the IMPACT analyzer with an IC100 of ~ 1 mcg/mL with citrate anticoagulation. In contrast to GPIIb/IIIa antagonists, ARC1779 did not inhibit platelet aggregation by ADP, collagen or arachidonic acid at concentrations (10mcg/mL) that fully inhibited vWF dependent platelet function. ARC1779 fully blocked vWF activity ex vivo with an IC90 of ~ 1 mcg/mL in young controls and 6 – 8 mcg/mL in STEMI and NSTEMI patients. Conclusions: ARC1779 potently and specifically inhibits vWF activity and vWF dependent platelet function, even in the setting of AMI where vWF activity is increased. ARC1779 represents a novel therapeutic principle (vWF antagonism) and a novel therapeutic class (aptamers). Potent and specific inhibition of VWF makes ARC1779 a promising development candidate for patients with AMI. Results

Reconfirmation of Preanalytical Variables and Establishment of Reference Intervals of Platelet Function Analyzer-100 Closure Times in Korean Adults

Platelet Function Analyzer-100 (PFA-100, Dade-Behring, Germany) is an instrument that simulates in vivo hemostatic plug formation under high shear flow by measuring the time required to occlude aperture. We performed this study to reconfirm preanalytical variables and establish the reference intervals of Korean adults. A total of 120 healthy individuals were enrolled. Closure times (CT) with the collagen/ epinephrine (CEPI) and the collagen/ADP (CADP) cartridges were measured. The reference intervals by the central 95th percentile were 82-182 sec for CEPI-CT and 62-109 sec for CADP-CT. Females had significantly longer CEPI- and CADP-CT than males (P=0.034 and 0.022, respectively). Individuals over 40 yr showed shorter CEPI- and CADP-CT compared with younger ones (P=0.002 and 0.003, respectively). CEPI- and CADP-CT values measured in the afternoon were significantly longer than corresponding ones in the morning (P<0.0001 in both conditions). Group O blood groups were related to longer CEPI- and CADP-CT compared with non-O blood groups (P=0.0003 and <0.0001, respectively). CADP-CT was weakly correlated with hematocrit (r=-0.296, P=0.001), but not CEPI-CT. We reconfirmed the preanalytical variables and established the reference intervals of PFA-100 CT in Korean adults. It is recommended that reference interval of this test should be verified according to age, diurnal variation, and ABO blood groups for optimal utilization.

Clopidogrel-Induced Platelet Inhibition Cannot Be Detected by the Platelet Function Analyzer-100 System in Stroke Patients

The administration of an adenosine diphosphate (ADP) receptor antagonist, such as clopidogrel, is recommended for recurrent stroke patients under aspirin treatment. However, up to 25% of vascular patients have an inadequate response to clopidogrel treatment, which could be associated with increased reinfarction rates. This study investigated whether the platelet function analyzer (PFA-100) system represents an appropriate tool for monitoring clopidogrel's antiplatelet effects in stroke patients. Sixteen stroke patients on clopidogrel therapy (75 mg/day) were included in a prospective analyst-blinded, cross-sectional study. Platelet function was assayed by collagen/epinephrine (CEPI)- and collagen/ADP (CADP)-induced closure times (CTs) using the PFA-100 system. von Willebrand factor antigen (vWF-Ag) levels were measured by enzyme immunoassay. CEPI-CT and CADP-CT values averaged 160 +/- 15 seconds and 102 +/- 10 seconds, respectively, and were in the normal range. vWF-Ag concentrations averaged 153 +/- 17% and correlated inversely with CTs (r = .71; P < .002 for CEPI-CT, r = .54; P < .04 for CADP-CT). Our data indicate that the current PFA-100 cartridges are not sufficiently sensitive to detect clopidogrel-induced platelet inhibition in stroke patients.

Response to aspirin and clopidogrel in patients scheduled to undergo cardiovascular surgery

Recent data indicate that among patients undergoing percutaneous coronary intervention low platelet response to aspirin is associated with clopidogrel low response. It is unclear whether these findings extend to other patient populations. We, therefore, aimed to evaluate the relation between response to aspirin and clopidogrel among patients scheduled to undergo cardiac or vascular surgery. Patients who were scheduled for cardiac or vascular surgery and had taken aspirin 81-325 mg daily for at least a week and clopidogrel 75 mg daily for at least 3 days underwent blood testing for platelet function. One hundred patients were included in the current analysis. Platelet function was evaluated by the modified TEG platelet mapping assay with addition of ADP or arachidonic acid (AA), and by the PFA-100 assay with collagen-epinephrine (CEPI) or collagen-ADP (CADP) cartridges. Low response to aspirin or clopidogrel was defined as inhibition < or =20% for TEG-AA or TEG-ADP, respectively. Thirteen patients (13%) were low responders to aspirin and 34 (34%) were low responders to clopidogrel. Eight patients were low responders to both drugs. There were no differences in clinical characteristics between drug low responders versus sensitive patients. Aspirin low responders had lower TEG-ADP inhibition (19.5 +/- 6 vs. 35.8 +/- 3%, P = 0.03) and tended to have lower PFA-CADP time (84.7 +/- 7 vs. 105.6 +/- 5 s, P = 0.1) than aspirin sensitive patients. Clopidogrel low responders had lower TEG-AA inhibition (58 +/- 6 vs. 75.1 +/- 4%, P = 0.01) and PFA-CEPI time (168 +/- 13 vs. 200.4 +/- 10 s, P = 0.07) than clopidogrel sensitive patients. In patients scheduled to undergo cardiovascular surgery low response to aspirin is associated with low response to clopidogrel.

Effects on primary haemostasis of an anti-inflammatory agent with 5-lipoxygenase and cyclooxygenase inhibitory activity

Licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) has been demonstrated to inhibit cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase. The aim of this study was to investigate the in-vitro effects of licofelone on platelet function. Effects observed were compared with those produced by the classic COX-1 inhibitor aspirin (ASA). Platelet aggregation was assessed by a turbidimetric method. Platelet haemostatic performance was studied with the platelet function analyser (PFA-100), using collagen epinephrine and collagen ADP cartridges. Interaction of platelets with thrombogenic surfaces was analysed by perfusion experiments performed under flow conditions using both parallel and annular chambers. Licofelone prolonged the lag time of platelet aggregation induced by arachidonic acid and reduced maximal platelet aggregation induced by ADP or collagen. Studies using PFA-100 demonstrated that licofelone (0.1, 1 and 10 muM) significantly prolonged closure times (P < 0.05) with both types of cartridges. In studies with the parallel chamber exposing purified collagen, both licofelone and ASA significantly reduced (P < 0.05) overall platelet interaction with the thrombogenic surface. In studies performed in annular chamber exposing a highly thrombogenic vessel surface, licofelone reduced height and area of the platelet masses deposited (7.0 +/- 0.5 mum; P < 0.005 and 80.2 +/- 17.3 mum; P < 0.05 vs. control 10.6 +/- 0.9 mum and 194.8 +/- 44.7 mum, respectively). ASA also impaired thrombus formation but differences did not reach the levels of statistical significance. Under our experimental in-vitro conditions, licofelone interfered with platelet function as demonstrated by a diminished platelet aggregation, being more powerful than ASA and reducing the interaction of platelets with thrombogenic surfaces.

Platelet activation predicts recurrent ischemic events after percutaneous coronary angioplasty: A 6 months prospective study

Introduction An increasing amount of evidence indicates that platelet reactivity, despite a standard anti-thrombotic therapy, is a potential risk factor for recurrent myocardial ischemia in patients with coronary artery disease. We now hypothesize that this condition, measured by collagen–epinephrine (CEPI) or collagen–ADP (CADP) closure times (CT) by Platelet Function Analyzer (PFA-100), may predict the recurrence of coronary events after percutaneous coronary intervention (PCI). Materials and methods CEPI and CADP-CT were measured 30 ± 8 h after PCI in 175 consecutive patients admitted with a diagnosis of stable angina ( n = 94) or acute coronary syndromes ( n = 81) and prospectively followed up for a mean period of 6 months. We stratified the patients in accordance to both the CEPI-CT (<or> 190 s), reflecting the intensity of cycloxygenase inhibition by aspirin and the distribution into quartiles for CADP-CT. Results CEPI-CT < 190 s as well as CADP-CT < 82 s were associated with a higher rate of clinical recurrence (hazard ratio 8.5, p < 0.001 and 22.9, p < 0.001, respectively). Multivariate analysis after adjustment for other risk factors confirmed that the lowest CADP-CT quartile significantly correlates with the risk of recurrent coronary events (hazard ratio 36.5, p < 0.01), as well as CEPI-CT < 190 s (hazard ratio 6.7, p = 0.01). Conclusions An enhanced platelet function after PCI when measured under high shear rates by PFA-100 is an independent predictor of a worst clinical outcome, even during a short term follow-up and may help in patients risk stratification.

Variables influencing Platelet Function Analyzer‐100TM closure times in healthy individuals

We investigated the relationship between platelet function analyzer (PFA-100) closure times (CT) and bleeding time (BT), platelet aggregation (PA) induced by ADP, arachidonic acid, and collagen, blood cell counts, and von Willebrand factor (VWF) in 120 well-characterised healthy individuals. Pre-analytical and analytical conditions were standardised comprehensively. In a substantial number of cases the differences between duplicate measurements exceeded 15%. The reference range (5th and 95th percentiles) for CT with the collagen/epinephrine (CEPI) and the collagen/ADP (CADP) cartridge was 93-223 s and 64-117 s respectively. Re-examination of 11 individuals with CEPI-CT above the 95th percentile revealed considerable batch-to-batch variation of CEPI-CT. Males had significantly longer CADP than females (P = 0.002). CEPI and CADP-CT measured pm were significantly longer than corresponding values determined am (P = 0.003 and P < 0.0001 respectively). Blood group O was associated with greater CEPI and CADP-CT and lower VWF levels compared with non-O blood groups (P = 0.008, P = 0.0003 and P < 0.0001 respectively). Linear regression analysis revealed association between CEPI-CT, CADP-CT and VWF (P < 0.0001), but no relationship was found between CT and BT or between CT and PA. We conclude that VWF plasma levels modulate PFA-100 CT to a greater extent than platelet function. Establishment of reliable reference ranges and careful standardisation of pre-analytical and analytical conditions is a prerequisite for obtaining reliable PFA-100 results. Duplicate measurements are necessary.

Laboratory Analysis of Abnormal Closure Times with Relation to Low Ristocetin Cofactor Versus Other Causes.

Closure times (CTs) have replaced the limitations of the previous testing methods. However, the clinical utility of detecting abnormal CTs remains poorly defined, with the differential diagnosis including von Willebrand's disease (vWD), aspirin or NSAIDs, uremia, liver disease, or congenital defects. Ristocetin cofactor (RCoF) measures the functional activity of the vW molecule and when low, most often suggests vWD. CTs are frequently done in patients suspected by history of having vWD with some laboratories preferring to perform the collagen/epinephrine (Col/Epi) CT alone, while others additionally include the collagen/ADP (Col/ADP) CT.METHODS: We analyzed 1480 consecutive CTs from a large reference laboratory done on a PFA-100 (Dade-Behring) and correlated the CTs with RCoF levels done on a BCS analyzer (Dade-Behring). Normal ranges were: Col/Epi CT 84-175 sec.; Col/ADP CT 65-117 sec.; RCoF 0.5-1.5 U/ml. Calculated means for CT of ≥ 300 sec. used a level of 300.TABLEOf the 260 studies with abnormal closure times, 131 (50%) had normal RCoF values, 47 (18%) were high and 18 (32%) were low. All studies with low RCoFs had at least one abnormal CT with 78/82 (95%) having both CTs prolonged, 2/82 (≫2%) with a long Col/EPI only, and 2/82 (≫2%) with a long Col/ADP only. Mean CTs in patients with low RCoFs and both CTs abnormal were 252 seconds for Col/EPI and 174 seconds for Col/ADP, both well above the upper limits of the normal range. Mean CTs in patients with low RCoFs and one abnormal CT were 180 seconds for Col/EPI and 123 seconds for Col/ADP, both just slightly above the upper limits of the normal range.CONCLUSIONS: These findings suggest that when RCoF is low, both Col/EPI and Col/ADP CTs are prolonged, usually markedly, and the chances that a low RCoF will be missed is low, particularly if both CTs are used.RESULTS: Total CTs analyzed: 1480 No.≥ 1 Abnormal CT 260/1480 (18%)Number (%)Both CTs AbnormalCol/Epi ONLY AbnormalCol/ADP ONLY AbnormalTotal Abnormal CTs260 (100%)203 (78%)42 (16%)15 (6%)Total Low RCoFs82 (100%)78 (95%)2 (≈2%)2 (≈2%)

Assessment of a platelet function analyser in horses: Reference range and influence of a platelet aggregation inhibitor

The objective of this study was to assess whether a new human platelet function analyser (the PFA-100) could be used to evaluate platelet function in horses and detect acetylsalicylic acid (ASA)-induced platelet dysfunctions. Citrated blood samples from 40 healthy horses were processed to obtain reference values for closure time (CT) using cartridges with collagen–ADP (CT–ADP) and collagen–epinephrine (CT–EPI) as platelet agonists. In addition, CT–ADP and CT–EPI were also measured before and 24 h after oral ASA administration in another 12 healthy horses. The sensitivity and specificity of the test were also determined. In normal horses, means ± SD value for CT–ADP was 85.1 ± 13.1 s (median, 82 s), and CT–EPI ranged from 158 to >300 s (median 291 s). Calculated reference ranges were 60.5–115.9 s and 158.5–>300 s for CT–ADP and CT–EPI, respectively. Administration of ASA significantly ( P < 0.001) prolonged CT–ADP values from 91.0 ± 13 to 113.5 ± 14.4 s, and CT–EPI values were also significantly ( P < 0.008) prolonged after ASA administration. Sensitivity and specificity results for ADP cartridges showed that a prolonged CT value would be highly suggestive of a platelet aggregation inhibition. In conclusion, ADP cartridges can be used in horses to assess primary haemostasis and may be a valuable test for the detection of platelet aggregation inhibition.

High incidence of defective high-shear platelet function among platelet donors.

Because single-donor plateletpheresis concentrates now account for a large percentage of PLT concentrates, the PLT quality of individual donations is increasingly important. There has been little previous work on PLT function in blood donors. The aim of this study was to investigate the prevalence of defective PLT function among 100 healthy UK PLT donors on 3 consecutive donation days. Citrated blood samples were taken before plateletpheresis. High-shear PLT function testing was performed by a PLT function analyzer (PFA-100, Dade Behring), within both collagen-ADP (CADP) and collagen-epinephrine (CEPI) cartridges. Sixteen percent of PLT donors (mainly over the 3 donating days) had prolonged CEPI closure times (CTs) only, indicative of an aspirin-like pattern; 9 percent had a severe defect with prolonged CEPI CTs between 200 and 300 seconds, and 4 percent had maximal CEPI CTs of greater than 300 seconds. The majority of prolonged CEPI CTs appeared transient in nature. PLT dysfunction as detected by the PFA-100 is surprisingly common within a voluntary PLT donor population. The majority of CEPI defects appeared transient in nature and are indicative of surreptitious intake of cyclooxygenase inhibitors (e.g., aspirin). Identification of defective PLT function within donors would provide a simple means to eradicate defective PLTs from being clinically utilized.

The effects of flavanol-rich cocoa and aspirin on ex vivo platelet function

Background: Flavanols modulate platelet function in vitro, but less is known of their in vivo effects and how they compare to pharmacological platelet inhibitors. We investigated the effect of a flavanol-rich cocoa beverage (897 mg/ml) in combination with and in comparison to aspirin on platelet function and activation in healthy subjects. Methods and results: On separate test days in a crossover design, 16 healthy adults consumed aspirin (81 mg), cocoa (as a beverage), or aspirin plus cocoa. Platelet activation was measured by surface expression of P-selectin and PAC-1 binding to the activated conformation of the GPIIb/IIIa receptor (GPIIb/IIIa-act). Platelet function was measured on an analyzer (the PFA-100™) that measures shear stress-induced platelet plug formation in response to collagen–epinephrine or collagen–ADP. Plasma epicatechin concentrations peaked approximately 2 h after subjects were given either the cocoa or aspirin plus cocoa. After 6 h, cocoa inhibited epinephrine-induced platelet function. Epinephrine-induced platelet function was inhibited 2 and 6 h after aspirin, and after aspirin plus cocoa. Epinephrine-stimulated P-selectin expression was inhibited by aspirin at 6 h, and after 2 and 6 h by aspirin plus cocoa. ADP-stimulated P-selectin expression was not affected by the treatments. Cocoa and aspirin, given separately, reduced epinephrine-stimulated GPIIb/IIIa-act expression at 2 and 6 h, respectively, and at 2 and 6 h when given together, suggesting an additive effective. ASA plus cocoa inhibited ADP-stimulated GPIIb/IIIa-act expression at 6 h. Conclusions: Flavanol-rich cocoa inhibited epinephrine-stimulated platelet activation and function. These effects were qualitatively similar to aspirin, but less profound. These results emphasize the need to further examine the effects of food flavonoids for platelet modulating effects.

Impact of plasmapheresis on platelet hemostatic capacity in healthy voluntary blood donors detected by the platelet function analyzer PFA-100™

Previous flow cytometry studies showed that platelet activation may occur during cytapheresis, cardiopulmonary bypass and hemodialysis. The aim of this pilot study was to determine if the impact of plasmapheresis leads to alterations in platelet hemostatic capacity. Plasmapheresis was carried out in 30 volunteers using an Autopherese C™ (Baxter) and an MCS3p™ (Haemonetics). Blood samples were collected and analyzed immediately before and after plasmapheresis. As a result of the plasmapheresis on the Autopherese C™, the mean closure time (CT) for collagen/epinephrine (Col/Epi) increased from 118.2 - 25.1 to 149.9 - 35.0 s and from 88.7 - 16.9 to 98.5 - 26.3 s for collagen/ADP (Col/ADP), respectively. Seven subjects showed impaired CTs after plasmapheresis for Col/Epi. For Col/ADP, five subjects showed impaired CTs after apheresis. Statistical analysis (McNemar test) showed a significant difference before and after apheresis for Col/Epi ( P = 0.024) but not for Col/ADP ( P = 0.088). Similar results were shown for plasmapheresis carried out with the MCS3p™. These findings show that plasmapheresis can cause an impairment of platelet function in healthy volunteers as measured by the PFA-100™, an effect of so far unknown clinical significance.

Assessment of Primary Hemostasis by PFA-100® Analysis in a Tertiary Care Center

We evaluated the utility of the PFA-100 platelet function analyzer in identifying disorders in platelet function and/or von Willebrand factor (vWF) in patients with various systemic disorders being followed at a tertiary care center. Closure times were determined with collagen/ ADP (CADP) and collagen/epinephrine (CEPI) cartridges for 305 patients, and abnormal results were further evaluated with platelet aggregometry and vWF analysis. Prolonged CADP and/or CEPI closure times were identified in 114 patients (37.3%), but most were isolated prolonged CEPI closure times predominantly due to aspirin therapy (79 patients). Prolonged CADP closure times were most frequently due to qualitative platelet defects and/or decreased vWF levels. Prolonged CADP closure times were encountered most frequently in patients with sickle cell disease and were associated with a decreased hematocrit. This study demonstrated that the PFA-100 analyzer can accurately assess vWF-dependent platelet function and detect other platelet defects under high shear stress in complex patient populations.

Standardization of the PFA-100(R) platelet function test in 105 mmol/l buffered citrate: effect of gender, smoking, and oral contraceptives.

The PFA-100(R) (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3.2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from five blood centres was confirmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges. PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut-off value for the Col/Epi CT was slightly higher for smokers than for non-smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of specific reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82-150 s; Col/ADP 62-100 s). Normal levels of fibrinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to influence the PHC. Because slight but significant differences of the reference ranges were observed between some of the participating sites, in-house confirmation of these reference range guidelines is recommended.

Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100

The PFA-100 is a new apparatus used to detect platelet dysfunction in vitro . Anticoagulated blood flows under constant pressure through a capillary, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP. Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet function. Using electron microscopy and immunogold staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and that are responsible for the occlusion. Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagenADP cartridges presented the same morphological features. The aggregates were exclusively composed of platelets, some of which were degranulated. Degranulation was particularly intense at the periphery of the aggregate where platelets were often totally devoid of secretory organelles. Immunogold staining on ultrathin frozen sections with polyclonal antibodies, allowed us to evaluate the distribution of adhesive proteins such as fibrinogen and von Willebrand factor (vWF) within the aggregate. The latter was found to be abundant in the intercellular spaces between adjoining platelets. Although fibrinogen was also present, its labeling was less intense suggesting that vWF is the major protein implicated in the platelet-platelet interactions in the aggregates formed in the PFA-100 system. This may be because of the high shear rate that occurs across the aperture which suggests that the PFA-100 is particularly sensitive for detecting abnormalities of vWFplatelet interactions.

COAGULATION STUDIES ON PATIENTS WITH SICKLE CELL DISEASES

Repeated measurements of coagulation factor activities were done on blood from 16 patients with Sickle Cell Disease over a one year period. Sixty-two coagulation profiles were available for analysis during well periods and 12 profiles on 6 patients were obtained during painful crisis. The profiles included measurements of the prothrombin time, partial thromboplastin time, thrombin time, platelet count, quantitation of Factors I, II, V, VII, VIII, IX, X, XI, XII, measurement of fibrin split products, fibrin monomer, and platelet aggregation with collagen ADP and epinephrine. Factor II was significantly lower than normal, platelet counts were significantly increased, and platelet aggregation with collagen epinephrine and ADP were impaired in approximately one-third of patients in. the absence of known injestion of platelet active drugs. Fibrin monomer as measured by radioactive amine incorporation (JCI 50:2235, 1971) was increased once in each of 4 patients but there were no increased levels of fibrin split products in the serum and no significant differences in results during well and painful crisis periods. Therefore, no evidence of chronic intravascular coagulation was found. The decreased concentration of prothrombin may be a result of liver impairment. The increased platelets and abnormal aggregation are unexplained.