Plasma fibronectin promotes thrombus growth and stability in injured arterioles

Abstract

Mice lacking both of the best-known platelet ligands, von Willebrand factor and fibrinogen, can still form occlusive thrombi in injured arterioles. The platelets of these animals accumulate excessive amounts of fibronectin (FN). These observations led us to examine the contribution of plasma FN (pFN) to thrombus formation. Inactivation of the FN gene in FN conditional knockout mice reduced pFN levels to <2% and platelet FN to approximately 20% of the levels in similarly treated control mice. The mice were then observed in a model of arterial injury to evaluate their capacity to form thrombi. The deficiency of pFN did not affect the initial platelet adhesion, but a delay of several minutes in thrombus formation was observed in the arterioles of pFN-deficient mice as compared with control mice. The thrombi that formed in the absence of pFN were stably anchored to the vessel wall but continuously shed platelets or small platelet clumps, thus slowing their growth significantly; the platelet/platelet cohesion was apparently diminished. Consequently the occlusion of pFN-deficient vessels was delayed, with the majority of vessels remaining patent at the end of the 40-min observation period. We conclude that, in addition to von Willebrand factor and fibrinogen, FN plays a significant role in thrombus initiation, growth, and stability at arterial shear rates and that deficiency in each of the three platelet ligands has its own specific impact on platelet plug formation.

Figure 1

Determination of FN levels in plasma of FN^flox/flox mice after treatment with polyI-polyC. Shown are two dilutions of plasma from two pairs of young recipient mice with or without the Mx-cre transgene, compared with a serial dilution of plasma from WT mice (Right) untreated with polyI-polyC. Note that the cre⁻ mice are indistinguishable from WT, whereas the cre⁺ mice showed very low levels of FN. Quantitation of such gels showed that the signal in 1:50 dilution of WT plasma was stronger than that of undiluted cre⁺ samples. Thus the cre⁺ mice had levels of fibronection <2% of WT.

Figure 2

Thrombus growth in the presence or absence of pFN. Times after FeCl₃-induced injury are indicated in bold. In the control mouse (Upper) and the pFN-deficient mouse (Lower), single fluorescent platelets are seen to adhere in the arterioles at 3 min after injury. Whereas in the control vessel large stable thrombi grew (15 min), leading to occlusion at 22 min, only very small thrombi were apparent at 15 min in the pFN-deficient animal, and at termination of the experiment (40 min) the vessel was still patent. The lack of stability of the thrombus formed in the pFN-deficient mouse is better observed in Movie 1, where the two arterial segments are shown in real time.

Figure 3

Quantitative analysis of formation of thrombi in control (black bar) and pFN-deficient (striated bar) arterioles. (A) Initial platelet deposition. The number of fluorescent platelets deposited per min was determined in the interval of 3–5 min after injury. pFN appeared not to influence significantly the early platelet interactions with the subendothelium. (B) Time required for the formation of the first thrombus >20 μm in diameter. The absence of pFN significantly delayed the formation of thrombi. (C) Embolization. The number of large emboli >30 μm generated in the period before occlusion was determined. Although in the absence of pFN the thrombi were dissolving by shedding single or small groups of platelets, formation of large emboli was just as infrequent as in animals with WT levels of pFN.

Figure 4

The role of pFN in occlusion of injured arterioles by platelet thrombi. The time needed to form an occlusive thrombus in an injured arteriole was determined in 9 control and 12 pFN-deficient animals. Occlusion was noted when blood flow completely stopped for at least 10 s. If this did not occur during the observation period of 40 min, 40 min was taken as the occlusion time. The majority of vessels did not occlude in pFN-deficient mice, whereas this happened in only two control animals. Thus there was a significant defect in generation of occlusive thrombi in the absence of pFN. P < 0.009.