Abstract
Background: A precise approach to the diagnosis of von Willebrand disease (vWD) remains elusive. One important reason is that vWD is a blood flow‐related disorder: a von Willebrand factor (vWF)‐platelet GPIb binding defect exists in this condition under the high shear‐rate (>1000 sec‐1 in whole blood; >3000 sec‐1 in PRP) conditions of physiologic blood flow which exist in the arterioles of mucous membranes, from which most bleeding in vWD occurs.
Objectives: The aims of this work are 1) to differentiate patients with true type 1 von Willebrand disease or symptomatic low vWF from normal controls and those with asymptomatic low vWF, as some patients with low vWF have a bleeding phenotype; and 2) to demonstrate the feasibility of a microfluidic device to so differentiate at a shear rate of 4000 sec-1 and with a minimal blood volume.
Methods: We studied in replicate 10 patients (mean 19.6 yrs) with type 1 vWD, diagnosed according to the 2007 NHLBI clinical guidelines, 4 other subjects considered to have type 1 vWD based on low levels of the vWF, and 8 healthy controls (mean 22.4 yrs). The PDQ bleeding assessment tool was applied to all subjects with or considered to have type 1 vWD. Blood was collected into a plastic tube containing 4 U/ml FC enoxaparin, 1.75 μg/ml of the Tab (anti‐CD41) monoclonal antibody directed against platelet GPIIb, and 1.0 μg/ml of an ALEXA 555‐conjugated rabbit anti‐mouse secondary antibody. Within 30-90 min, the blood was withdrawn at 4,000 sec‐1 through a special flow chamber (Fig. 1) allowing for real-time epifluorescence digital videomicroscopy of platelets interacting with a microfibrillar collagen substrate. With MetaMorph software (Universal Imaging) we quantified the percent area (PA) covered by adherent platelet aggregates (Fig. 2) within a 435 μm x 580 μm field of view (1,447,680 pixels). From values for PA vs. time (Fig. 3) we also calculated the rate (R) of platelet adhesion. In some studies, we imaged fibrin deposition vs. time, using the monoclonal antibody 59D8, which is directed against human fibrin β-chain, and a fluorescent secondary antibody.
Results: As shown in Fig. 4, at 4,000 sec-1 R for normal controls was 2.1-fold (p < 0.00001) greater than that for patients (Group I), while from previous work we found no difference at 667 sec-1 and only a 1.4-fold difference at 1330 sec-1. PA at 2 min for controls increased with shear rate from 7.5 at 667 sec-1 and 12.8 at 1,330 sec-1, but then decreased to 8.6 at 4,000 sec-1. In contrast, PA for patients (same units) increased from 8.0 at 667 sec-1 to only 9.5 at 1330 sec-1, and then decreased to 5.3 at 4,000 sec-1. Importantly, all ten patients had values for R that fell below the lower 95th percentile for controls. Four other subjects (Group II), considered to have type 1 disease, proved to have bleeding scores that were ≤ 2. One of these was an adult with absolutely no personal bleeding history despite a family history of vWD. Values for PA and R for these subjects were similar to values for normal subjects. For normal controls and Group I patients, at 270 sec-1, fibrin deposition lagged platelet adhesion/aggregation by 5-7 min, but then occurred in a burst in the slow-flow wakes of platelet aggregates as well as on (activated) platelet membranes. At 4000 sec-1, fibrin deposition, developed within 30 sec and was confined to platelet membranes for both normal controls and Group I patients. Group II subjects were not studied.
Conclusions: The novel flow device found that PA and rate of platelet adhesion were significantly reduced under high shear stress in "true" type 1 vWD patients and those with symptomatic low vWF compared to normal controls and those with asymptomatic low vWF. This likely results from the "catch-slip" nature of bonds between the vWF and either platelet GPIb or collagen, which has been well-described for the vWF-GPIb interaction with regard to platelet suspensions. Fibrin deposition occurred on (and/or beneath) forming platelet adhesion/aggregation and was in proportion to the level of platelet adhesion/aggregation. The present approach promises to offer a more definitive means to distinguish patients with true type 1 vWD or those with symptomatic low vWF from either normal controls or those with symptomatic low vWF.
Grabowski: CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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