Differential Roles of Fibrinogen and von Willebrand Factor on Clot Formation and Platelet Adhesion in Reconstituted and Immune Thrombocytopenia Mudi Misgav, MD,* Boris Shenkman, MD, PhD,* Ivan Budnik, MD,* Yulia Einav, PhD,† and Uri Martinowitz, MD* BACKGROUND: Bleeding tendencies in immune thrombocytopenia (ITP) do not always correlate with the number of platelets, suggesting platelet function variation. We used a model of normal whole blood thrombocytopenia to compare platelet function and other hemostatic variables with ITP patients. We further investigated the effect of in vitro spiking with von Willebrand factor (vWF) and fibrinogen on platelet function and hemostatic variables. METHODS: The Cone and Plate(let) Analyzer was used to measure platelet adhesion (surface coverage [SC], %) and aggregation (average size, m 2 ) under defined shear rate (1200 s -1 ). Rotational thromboelastometry was used to determine variables of clot formation triggered by CaCl 2 and tissue factor. RESULTS: In both the model of thrombocytopenia as well as in ITP, the SC and to some extent the average size were correlated to the platelet number over a range of 5 to 80  10 6 /mL. The results obtained for most ITP samples were within the boundaries of the lower and upper limits set by the whole blood model of thrombocytopenia. The addition of 2 U/mL vWF (Haemate-P) to whole blood (calculated to plasma volume) results in an increase in the SC and average size without affecting clot formation. Spiking with fibrinogen (100 and 300 mg/dL) did not affect platelet deposition but improved clot formation. CONCLUSIONS: Using a model of whole blood thrombocytopenia enables us to establish reference variables for the Cone and Plate(let) Analyzer and rotational thromboelastometry and to assess platelet function and clot formation in the presence of severe thrombocytopenia. We demonstrated that in most cases of ITP, platelet function is comparable to normal platelets. This work also suggests that vWF and fibrinogen differentially affect primary and secondary hemosta- sis and therefore both may perform a function in the bleeding phenotype and possibly may be considered for treatment in patients with ITP. (Anesth Analg 2011;112:1034 –40) I mmune thrombocytopenia (ITP) is an autoimmune disorder that is characterized by low platelet count due to accelerated platelet destruction as a result of autoan- tibodies against platelet surface antigens and T-cell toxic- ity. 1–3 Occasionally, impaired megakaryocytopoiesis has been found. 4,5 The consequence of thrombocytopenia (TCP) is complex because platelets are important not only in building up the primary hemostatic plug but also for thrombin generation and clot retraction. 6,7 Bleeding symp- toms in ITP are variable, ranging from mild to severe and occasionally may even be life-threatening. The conven- tional way to manage patients according to the degree of TCP does not include the assessment of platelet function. Bleeding is usually associated with a reduction of platelet count below 20  10 6 /mL. However, in some cases, bleed- ing is absent even at platelet counts of 10  10 6 /mL or lower. Conversely, bleeding may be present at platelet counts that exceed 30  10 6 /mL. These clinical observa- tions suggest the presence of platelet function diversity and that more defined ways to evaluate platelet function as well as strategies to improve management in case of severe bleeding are needed. Current expert consensus for first-line treatments includes the use of steroids, anti-D, and immune globulins. A response can take several days to weeks depending on the type of treatment, and side effects may be severe. 8 Also, a justified use of these treatments before invasive procedures is based on expert recommendations in regard to the platelet numbers only. 9 For the purpose of platelet function evaluation, neither platelet aggregation studies nor standard clotting assays may serve for comprehensive platelet function and clot formation assessment in ITP. Of note, light transmittance platelet aggregometry, the “gold standard” method of platelet function assessment, cannot be used when the platelet count is 100  10 6 /mL. 10 Moreover, platelet deposition in whole blood (WB) under defined shear stress is much more relevant for platelet studies approximate to blood flow in arterial vessel walls. The Cone and Plate(let) Analyzer (CPA; DiaMed Inc., Crefier, Switzerland) has been developed and has proved to be useful for the assessment of platelet function and for monitoring of antiplatelet and replacement therapy. 11–14 In the last decade, the rotational thromboelastometry (ROTEM) has emerged as a valuable point-of-care device and a research tool for the assessment and monitoring of hemostasis and fibrinolysis. 15,16 In that respect, ROTEM From the *Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; †Holon Institute of Technology, Holon, Israel. Accepted for publication January 10, 2011. The authors declare no conflicts of interest. Reprints will not be available from the authors. Address correspondence to Boris Shenkman, MD, PhD, Institute of Thrombosis and Hemostasis and National Hemophilia Center, the Chaim Sheba Medical Center, Tel Hashomer, 52621, Israel. Address e-mail to borshenk@sheba.health.gov.il. Copyright © 2011 International Anesthesia Research Society DOI: 10.1213/ANE.0b013e318212fffc 1034 www.anesthesia-analgesia.org May 2011 • Volume 112 • Number 5