Hematocrit and flow rate regulate the adhesion of platelets to von Willebrand factor

Primary hemostasis and blood clotting is known to be influenced by the red blood cell volume fraction (hematocrit) in blood. Depressed or elevated levels of red blood cells can lead to vascular perfusion problems ranging from bleeding to thrombus formation. The early stage of hemostasis and thus blood clotting in all vessel sections from the arterial to the venous system involves the adhesion of platelets to von Willebrand factor. Here we present experimental and theoretical results showing that the adhesion probability of platelets to von Willebrand factor is strongly and nonlinearly dependent on hematocrit and flow rate. Interestingly, the actual binding forces are not markedly different, which suggest that the origin of such behavior is in the distribution of platelets. Using hydrodynamic simulations of a simple model, we explicitly show that the higher the hematocrit and the flow rate, the larger the amount of platelets residing close to the wall. Our simulation results, which are in excellent agreement with the experimental observations, explain why such phenomena occur. We believe that the nonhomogeneous red blood cell distribution as well as the shear dependent hydrodynamic interaction is key for the accumulation of platelets on the vessel wall. The work we present here is an important step forward from our earlier work on single molecules and extends into the collective cellular behavior of whole blood. It sheds new light on the correlation between hematocrit and the initial steps in hemostasis and thrombosis, and outlines advances for the treatment of vascular diseases associated with high levels of red blood cells. These results are not only highly relevant for the field of hemostasis and the physics of blood clotting but are also of powerful impact in applied science most obviously in drug delivery and colloidal science.     

Biological variations of ADAMTS13 and von Willebrand factor in human adults

Background:

The ultra-large von Willebrand factor (vWF) multimers are very active and must be degraded by ADAMTS13 for optimal activity. A severe functional deficiency of ADAMTS13 has been associated with thrombotic thrombocytopenic purpura. The correct interpretation of patient vWF and ADAMTS13 plasma levels requires an understanding of the biological variation associated with these analytes. In the present paper, we aimed to determine the biological variation of ADAMTS13 and vWF in human adults.

Materials and methods:

Blood samples were collected weekly from 19 healthy subjects for 5 consecutive weeks. vWF activity and antigenicity were determined using aggregometric and immunoturbidimetric methods. ADAMTS13 antigenicity and activity were determined by ELISA.

Results:

The within-subject biological variations for vWF activity and antigenicity were 8.06% and 14.37%, respectively, while the between-subject biological variations were 18.5% and 22.59%, respectively. The index of individuality for vWF activity was 0.44, while vWF antigenicity was 0.64. Similarly, ADAMTS13 activity and antigenicity within-subject biological variations were 12.73% and 9.75%, respectively, while between-subject biological variations were 9.63% and 6.28%, respectively. The ADAMTS13 indexes of individuality were 1.32 and 1.55, respectively.

Conclusion:

We report high biological variation and individuality in vWF antigenicity and activity levels. However, ADAMTS13 antigenicity and activity displayed high biological variation, but low individuality. Thus, population-based reference intervals may be useful for monitoring ADAMTS13 antigenicity and activity, but not for vWF, which displays high individuality. These findings should be considered when determining the reference interval and other clinical variables associated with ADAMTS13 and vWF levels.     

Plasma centrifugation does not influence thrombin-antithrombin and plasmin-antiplasmin levels but determines platelet microparticles count

Introduction

Centrifugation is an essential step for plasma preparation to remove residual elements in plasma, especially platelets and platelet-derived microparticles (PMPs). Our working hypothesis was that centrifugation as a preanalytical step may influence some coagulation parameters.

Materials and methods

Healthy young men were recruited (N = 17). For centrifugation, two protocols were applied: (A) the first centrifugation at 2500 x g for 15 min and (B) at 2500 x g for 20 min at room temperature with a light brake. In protocol (A), the second centrifugation was carried out at 2500 x g for 15 min, whereas in protocol (B), the second centrifugation involved a 10 min spin at 13,000 x g. Thrombin-antithrombin (TAT) and plasmin-antiplasmin (PAP) complexes concentrations were determined by enzyme-linked immunosorbent assays. PMPs were stained with CD41 antibody and annexin V, and analyzed by flow cytometry method. Procoagulant activity was assayed by the Calibrated Automated Thrombogram method as a slope of thrombin formation (CAT velocity).

Results

Median TAT and PAP concentrations did not differ between the centrifugation protocols. The high speed centrifugation reduced the median (IQR) PMP count in plasma from 1291 (841-1975) to 573 (391-1010) PMP/µL (P = 0.001), and CAT velocity from 2.01 (1.31-2.88) to 0.97 (0.82-1.73) nM/min (P = 0.049). Spearman’s rank correlation analysis showed correlation between TAT and PMPs in the protocol A plasma which was (rho = 0.52, P < 0.050) and between PMPs and CAT for protocol A (rho = 0.74, P < 0.050) and protocol B (rho = 0.78, P < 0.050).

Conclusion

Centrifugation protocols do not influence the markers of plasminogen (PAP) and thrombin (TAT) generation but they do affect the PMP count and procoagulant activity.Key words: cell-derived microparticles, blood coagulation tests, centrifugation, preanalytical phase