Our results clearly confirm molecular heterogeneity in GPS. Previously, we have shown that patient 1 associates decreased platelet aggregation to collagen with an absence of GPVI (12
). Now we show that platelets of this patient are also severely deficient in another membrane glycoprotein, TLT-1. TLT-1 is a relatively little-studied membrane glycoprotein of human platelets, and belongs to the TREM receptor family (1
). Expression of TLT-1 is restricted to the megakaryocyte lineage and, typical of the TREM family contains an Ig superfamily V-type domain. TLT-1 is present as two isoforms that differ in their cytoplasmic domain. The larger form of TLT-1 contains two concensus ITIMs, potential sites for the recruitment of SH-domain-containing tyrosine phosphatases (3
). It is this isoform that is primarily recognized by our Western blot assay.
Previous confocal microscopy studies in two laboratories have shown that in normal platelets TLT-1 is colocalized with P-selectin, suggesting that this newly identified intrinsic membrane glycoprotein of platelets also has a granular localization (2
). Early I-EM studies by Israels et al showed that while P-selectin is primarily found in the membranes of α-granules, it also occurs in those of dense granules (13
). Our results clearly show that TLT-1 is principally associated with the α-granule membrane. Notwithstanding, some labelling was also found associated with smaller vesicular structures and occasionally on the platelet surface. As dense granules are not readily identifiable by the procedure used, the question as to whether the localization of TLT-1 extends to the dense granule membrane must remain open.
GPVI is a major platelet receptor for collagen (14
). Its virtual absence from the platelets of patient 1 is associated with a much decreased platelet response both to collagen and the GPVI-specific ligand, convulxin (12
). In contrast, platelets from patient 2 with a normal presence of GPVI, respond well to collagen (6
). Washington and his coworkers (2
) have shown that TLT-1 is a potential candidate to participate in the platelet aggregation response. This conclusion was based on the selective inhibition of thrombin-induced platelet aggregation using single-chain Fv antibodies specific for TLT-1. The same antibodies failed to inhibit collagen- and ADP-induced platelet aggregation, but surprisingly did inhibit that induced by the TXA2
analogue, U46619. One possibility is that these antibodies block the interaction between newly exposed TLT-1 and its purported (but non-identified) counter-ligand on adjacent platelets. An alternative theory put forward by Giomarelli et al (5
) is that TLT-1, through its ITIM domains, may regulate signalling pathways during platelet aggregation and SHP-sensitive Rho A-dependent activation was put forward as a candidate.
While for some GPS patients collagen-induced aggregation is the most severely affected, in others the defect primarily concerns the thrombin response (data reviewed in 6
). The basis for the altered thrombin response has never been explained. In a new case of GPS in Italy, a markedly reduced aggregation and Ca2+
-mobilization by thrombin was mimicked by peptides activating platelets through the PAR-1 and PAR-4 receptors (15
). An abnormal thrombin-induced Ca2+
-mobilization had been earlier reported for two French (16
) and an American patient (17
). It is interesting then that TLT-1 ligation has been reported to induce Ca2+
). Platelets from patient 1 also respond poorly to TRAP peptides acting through PAR-1 (see ref 12
); however, the decrease is not as severe as with collagen. As TLT-1 may play a role in secretion-dependent platelet aggregation together with secreted soluble α-granule proteins such as thrombospondin-1 (18
), it will be important to compare TLT-1 expression in a more extensive series of GPS patients.
GPVI deficiency in platelets is most often acquired and due to antibody-driven proteolysis (19
). Receptor loss through proteolytic activity by the so-called “sheddases” (a name given to a group of MMPs) is a natural way of down-regulating platelet function following ligand binding (21
). GPIbα, GPV, GPVI and P-selectin are among reported substrates in platelets. The fact that each of GPVI, TLT-1 and P-selectin were decreased in platelets of patient 1 reinforces the hypothesis that rogue MMP activity may be responsible and suggests that TLT-1 is also a potential target for cleavage. The fact that a soluble fragment of TLT-1 is found in human sera and in the supernatant after thrombin-induced normal platelet activation is compatible with it's being a target for MMPs (4
Platelets contain many MMPs including MMP-1, MMP-2 (gelatinase A), MMP-3, MMP-9 (gelatinase B), membrane-bound MT1-MMP, the VWF-cleaving protease ADAMTS-13, ADAM10 and tumor necrosis factor-α-converting enzyme (ADAM17) (21
). Our initial results showed the normal presence of MMP-2 and MMP-9 in platelets in GPS and although their proteolytic activity has not been measured, the α-granule deficiency was not accompanied by their loss suggesting other storage sites for these proteases. Principle candidates for cleaving GPVI are ADAM17 and especially ADAM10 (reviewed in 21
). The fact that GPIbα was normally present in the platelets of patient 1 means that the proteolysis is not generalized and further work is now required to establish the mechanism leading to selective GPVI, TLT-1 and P-selectin loss in the platelets of patient 1 and to establish the identity of the responsible protease(s) and the site where the cleavage occurs. In particular, the localization and activity of ADAM10 will need to be assessed.
In normal megakaryocytes, α-granule maturation passes by an intermediate step with the production of multivesicular bodies (31
). P-selectin is selectively sorted into the regulated secretory pathway by way of a recognition signal in the cytoplasmic domain (32
). Whether TLT-1 is likewise sorted will require further study. Some proteins are synthesized and packaged directly into the maturing α-granules in the megakaryocytes; PF4 is a well-studied example and, like P-selectin, has a granule targeting sequence (33
). In a second mechanism, proteins are captured by endocytosis and this is the case for Fg (34
). The presence of Fg in endocytic vesicles and/or precursor α-granules was clearly seen for platelets of patient 1 thereby confirming an earlier study (35
). When not cleaved, P-selectin and TLT-1 were mostly found lining vacuoles or elements of the OCS or even on the surface confirming that predestined α-granule membrane glycoproteins are redistributed in GPS platelets (7
Two other members of the Ig receptor family, JAM-C and claudin-5 were normally present in the platelets of both GPS patients. Unlike P-selectin and TLT-1, JAM-C was abundant on the surface of unstimulated platelets (see also refs 8
). To the best of our knowledge, claudin-5 has not been reported in platelets before. Neither JAM-C nor claudin-5 was identified as a target for proteolytic degradation in the GPS again confirming that receptor loss is selective. Whether the observed receptor loss occurs in the circulating blood or in the bone marrow during megakaryocyte maturation and/or platelet release remains to be shown. If the protease can be identified, then possible measures could be taken to inhibit this activity. The molecular defect(s) responsible for GPS remains elusive. Whether the phenotypic heterogeneity observed here is related to the different mode of inheritance in the two families is unknown. But it is tempting to suggest that more than one genetic defect can lie at the basis of the disease as is seen, for example, in the Hermansky-Pudlak syndrome, where multiple gene defects have been reported (36