Abnormal clot retraction, altered fibrin structure, and normal platelet function in multiple myeloma.

Clot retraction, measured by serum expression, is absent in some cases of multiple myeloma. Decreased clot retraction has been attributed to platelet dysfunction. A new instrument allows simultaneous measurement of platelet-mediated force development during clot retraction and of clot elastic modulus. We report 10 patients with immunoglobulin (Ig) G myeloma in whom the abnormalities of fibrin structure were quantitatively defined and platelet-fibrin interactions were assessed. Fiber mass-to-length ratios were calculated from gel turbidity. Platelet force development and clot elastic modula were measured in platelet-rich plasma gels. Fiber mass-to-length ratios for IgG myeloma patients were smaller (means +/- SE) (0.98 +/- 0.19 x 10(13) Da/cm) than for normal controls (1.36 +/- 0.06 x 10(13) Da/cm), indicating thinner fiber formation. Elastic modula of myeloma clots (51,013 +/- 14,660 dyn/cm2) were strikingly larger than modula for normal controls (23,355 +/- 1,887 dyn/cm2), indicating that such clots are mechanically less flexible. Platelet force development 1,200 s after thrombin addition was not diminished in myeloma patients (8,315 +/- 1,155 dyn) vs. controls (6,906 +/- 606 dyn). Abnormal clot retraction in myeloma appears to be primarily due to altered clot structure rather than platelet dysfunction.

Quantitative assessment of platelet function and clot structure in patients with severe coronary artery disease.

The prothrombotic state of patients with coronary artery disease (CAD) can be attributed partially to platelet activity. Management of such patients is hindered by a lack of techniques to assess hemostatic function. This study used a sensitive technique to monitor platelet function by measuring platelet force development during clot retraction. This technique allowed simultaneous measurement of clot elastic modulus on the same sample. Fibrin mass-length ratio (mu), fibrinopeptide A, D-Dimer, von Willebrand's factor, thromboxane A2, platelet aggregation studies, and bleeding times also were performed. Fourteen patients with CAD were compared with 10 healthy volunteers. Despite more than 95% suppression of thromboxane B2 and prolongation bleeding times in patients taking aspirin, force development remained significantly elevated over healthy control patients (8,279 +/- 476 dynes versus 4,857 +/- 380 dynes, p < 0.0006). Patients not taking aspirin had normal bleeding times and force development of 19,110 +/- 3,700 dynes. Clot elastic moduli were enhanced in patients with CAD whether taking or not taking aspirin. Adenosine diphosphate and ristocetin-induced platelet aggregation were insensitive to the effect of aspirin in patients with CAD. Fibrinopeptide A, von Willebrand's factor, and D-Dimer levels were significantly elevated, and fibrin mass-length ratios were significantly larger in patients with CAD. Therefore, despite aspirin therapy, patients with severe CAD have evidence of persistent platelet activation and rigid clot structure. Monitoring of platelet force development may prove useful in delineating enhanced platelet function.

Protein S levels during the normal menstrual cycle and during estrogen therapy for premature ovarian failure.

Protein S levels have been reported to be decreased in pregnancy and with oral contraceptive use. This study monitored the effects of estrogen shifts on protein S levels. Four patients with premature ovarian failure were treated with either oral or transdermal patch estrogen replacement. Blood drawn on days 1, 14, and 28 of therapy was analyzed for estradiol, estrone, free and total protein S, and C4b-binding protein (C4b-BP) levels. Similar studies were performed on six normally cycling control patients and seven postmenopausal women. In healthy females, total levels of protein S fell from 22.1 +/- 0.73 micrograms/mL on day 1 to 19.2 +/- 1.29 micrograms/mL on day 14 (p < 0.023). Free protein S levels declined from 6.45 +/- 0.70 micrograms/mL to 5.59 +/- 0.69 micrograms/mL (p < 0.016). C4b-BP levels did not change during the normal menstrual cycle. Baseline total protein S (44.1 +/- 7.0 micrograms/mL) and C4b-BP (193 +/- 18%) levels were elevated in patients with premature ovarian failure. On oral therapy, there was a strong, negative correlation (r = -0.979, p < 0.021) between C4b-BP and estradiol levels. C4b-BP levels did not change in patients with the patch. Both estrogen therapies produced similar declines (44 to 26 micrograms/mL) in total protein S levels. In all cases, total protein S levels changed as a reciprocal function of estradiol. C4b-BP (128 +/- 6.5%) and total protein S (32.2 +/- 3.0 micrograms/mL) levels were higher in postmenopausal women than in nonmenopausal females. Free protein S levels in postmenopausal women (9.6 +/- 0.6 micrograms/mL) were normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein S and C4b-binding protein levels in patients with stroke: implications for protein S regulation.

Protein S circulates either free or bound to C4b-binding protein (C4b-BP). Only free protein S possesses cofactor activity for protein C, a physiologic anticoagulant. Deficiencies of either protein C or protein S are associated with increased thrombotic risk. Over a 23-month period, 40 patients with low free protein S were identified. Eight of these patients were found to have suffered a stroke. This study examined the relationship between total S, free S, and C4b-BP in 15 healthy adult volunteers, in 20 patients with normal protein S levels, in 40 patients with decreased free protein S levels, and in 8 patients with combined low free S levels and stroke. Total and free protein S and C4b-BP levels were determined using the method of Laurell. In healthy adults, free protein S increased with increasing total protein S (r = 0.60). In patients with normal free S, the total S level increased as C4b-BP increased (r = 0.74), and the free S level remained constant. In patients with low free S, total S did not increase with increasing C4b-BP. In stroke patients, the correlation between free S and total S was actually negative (r = -0.449). Evaluation of dissociation constants for the protein S-C4b-BP complex revealed enhanced binding in patients with low levels of free protein S. A non-C4b-BP protein S binding protein, a previously undescribed regulatory factor which modulates S binding to C4b-BP, or shifts in the amount of non-protein S binding C4b-BP are possible explanations of these results.

Measurement of platelet-mediated force development during plasma clot formation.

We have developed an instrument that measures force development during clot retraction. Clots are formed between an aluminum plate and cup. The plate is coupled to a strain gauge transducer. Force generated by platelets is transmitted to the upper plate and the resulting voltage change is recorded. Calibration using standard weights allows conversion of the voltage signal to dynes/cm2. Study parameters include lag phase prior to force development, maximum rate of force development (MRFD), and maximum force generated (MFG). When controlled for platelet count, ionic strength (0.15), pH (7.4), calcium concentration (10mM), thrombin concentration (1.5 u/ml), temperature, and gap distance (1 mm), measurements are reproducible. Control of each variable is critical since clot retraction depends on platelet function and fibrin structure. At 37 degrees C and 26 x 10(3) platelets/microL, the lag phase was 300 seconds, the MRFD was 0.69 dynes/(cm2 sec), and the force at 1000 seconds (MFG) was 2450 dynes. At 7.5 x 10(4) platelets/microL, the lag phase was 160 seconds, the MRFD was 1.06 dyne/(cm2 sec), and the MFG was 4400 dynes. Decreasing the sample temperature from 37 degrees C to 27 degrees C (75 x 10(3) platelets/microL) increases the lag phase from 170 to 300 seconds, decreases MRFD from 1.06 to 0.66 dynes/(cm2 sec), and decreases the MFG at 1000 seconds from 4,400 to 2,800 dynes. At 15 degrees C, retraction is totally inhibited. This instrument provides a simple way to quantify the impact of altered fibrin structure and platelet function on clot retraction. Measurement of force development during clot retraction may offer a new approach to the study of qualitative platelet dysfunction.

Force monitoring of clot retraction during DDAVP therapy for the qualitative platelet disorder of uraemia: report of a case.

The qualitative platelet disorder of uraemia results in decreased primary haemostatic capacity which can result in significant blood loss during invasive procedures. Treatments of the disorder tend to be empirical and include measures such as aggressive dialysis, conjugated oestrogens, and use of DDAVP. Improvement in platelet function is typically monitored by repeated bleeding times. The variability of the bleeding time is an all too recognized limitation of its usefulness. We report here the case of a 35-year old male with end stage renal disease who presented with intractable bleeding secondary to peptic ulcer disease and haemorrhagic gastritis. Platelet function tests including bleeding time, platelet aggregation studies, and clot retraction measurements were monitored before and after intravenous administration of DDAVP (0.3 microgram/kg). The bleeding time which had been 8 min before DDAVP did not change. Platelet aggregation studies revealed improved aggregation by both collagen and adenosine diphosphate. Clot retraction forces were dramatically enhanced after DDAVP. Pre-DDAVP clots containing 72 x 10(9) platelets per 1 and 1 g fibrinogen per 1 produced 90 dynes/cm2 of force at 800 s post-thrombin addition. Identical clots formed with blood drawn 2 h post-DDAVP produced 750 dynes/cm2. The DDAVP dose was repeated after 8 h with obvious slowing of blood loss. The marked effect of DDAVP on clot retraction may allow monitoring of DDAVP therapy utilizing this technique.

Calculation of plasma fibrin fiber mass-length ratios utilizing platelet aggregometers.

We report a technique for measuring plasma gel fiber size utilizing optical platelet aggregometers. Three aggregometers were used to measure gelation kinetics and final gel turbidity: Sienco Model DP247E, Bio-Data Model PAP-2A, and Chrono-log Model 500-VS aggregometer. Each aggregometer was calibrated using dilutions of latex microspheres. Optical densities of microsphere solutions were measured at 626, 670 and 945 nm. Calibration curves were plots of aggregometer readings versus absorbance. Gels of various fiber size were prepared by varying thrombin concentrations and ionic strength. Fiber mass-length ratios were calculated from the wavelength dependence of gel turbidity. Gel optical densities at 640 nm and 945 nm were shown to be linear functions of fiber mass-length ratio. Aggregometer study gels were formed directly in aggregometer cuvettes. Gel formation kinetics were easily measured in the Sienco and Chrono-log instruments. Gelation kinetics in the Bio-Data instrument did not allow measurement of maximum turbidity. The latter value could be measured, however, once gelation was complete. Final aggregometer readings were made one hour after thrombin addition, and were converted to absorbance values using calibration curves. Absorbencies were then converted to mass-length ratios using the optical density versus mass-length ratio plots. Fiber mass-length ratios measured with aggregometers were in good agreement with those measured spectrophotometrically. This technique may allow routine quantification of plasma clot structure utilizing equipment ordinarily available in clinical laboratories.