Blood, 1 January 2002, Vol. 99, No. 1, pp. 387-388
CORRESPONDENCE
To the editor:
A growing set of platelet-activating bacterial proteins
We read with interest the paper by Lourbakos et al1
that reports activation of platelets by the Porphyromonas
gingivalis proteases HRgpA and RgpB, mediated through the
thrombin-responsive protease-activated receptors 1 and 4 (PAR1 and
PAR4). While this represents an interesting mechanism of platelet
activation by pathogenic bacteria, we feel the authors overlooked a
precedent for the paradigm of cellular activation through PARs by a
bacterial protein, that of streptokinase-induced, PAR1-mediated
platelet activation previously reported in
Blood.2
Unlike the P gingivalis-derived gingipains investigated by
Lourbakos et al, streptokinase (Sk) has no protease activity itself but
binds to plasminogen or plasmin, conferring new functions on the host
protein including the ability to activate plasminogen3 and
to functionally cleave PAR1 resulting in cell activation.2
Lourbakos et al show that HRgpA is a more potent agonist at
PAR4 than thrombin but less potent at PAR1. Thrombin is targeted both
to bind to PAR1 and to cleave it at the appropriate site by a
hirudinlike region adjacent to the cleavage site.4
However, other proteases reported to cleave PAR1 do so at multiple
sites resulting in a combination of receptor-activating and
-inactivating scissions.5 HRgpA activates human factors IX
and X, as well as prothrombin and protein C, suggesting a thrombinlike
substrate specificity.6 In all cases, this
activity is dependent on the non-covalently bound adhesin
subunit of HrgpA that may modify the relatively open binding site of
the gingipains.1
These data mirror our results with Sk. Sk alters the properties
of complexed plasmin, changing it from a protease that predominantly inactivates PAR1 by digestion at other sites5,7 into a
protease that activates the receptor by cleavage at the appropriate
site.2 Lourbakos et al speculate that the adhesin
component of HRgpA may also promote binding of that protease to the
platelet surface, facilitating PAR cleavage. Sk-induced platelet
activation also required an adhesive factor, provided by endogenous
anti-Sk antibodies (in plasma) binding to the platelet Fc
receptor.2
A similar binding pattern is seen in the presumed teleological
role of Sk: harnessing plasmin to allow bacterial invasion across
tissue barriers of new body compartments.8
Streptococci express binding sites on their surface for
plasminogen and fibrinogen, which form a ternary complex with Sk,
generating plasmin that remains cell-surface bound and escapes
inhibition by 
2-antiplasmin.8 Mammalian
cells also localize plasmin to their surface through the coexpression
of the urokinase plasminogen activator receptor (uPAR) and binding
sites for plasminogen, leading, for instance, to plasmin generation at
the leading edge of fibroblasts during wound healing.9
Indeed, on the mouse platelet, thrombin is targeted to PAR4 by the
hirudinlike region of PAR3.10 Thus many proteases are
regulated through colocalization with their activators or cofactors at
cell surfaces where they evade soluble inhibitors. This may contribute
to thrombosis when platelets' cell-surface protease-activated
receptors are cleaved by gingipains1 or Sk-plasmin.2
James P. McRedmond and Desmond J. Fitzgerald
Correspondence: Desmond J. Fitzgerald, Department of Clinical
Pharmacology, Royal College of Surgeons in Ireland, 123 St Stephen's
Green, Dublin 2, Ireland; e-mail: dfitzgerald{at}rcsi.ie
References
1.
Lourbakos A, Yuan YP, Jenkins AL, et al.
Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity.
Blood.
2001;97:3790-3797[Abstract/Free Full Text].
2.
McRedmond JP, Harriott P, Walker B, Fitzgerald DJ.
Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1.
Blood.
2000;95:1301-1308[Abstract/Free Full Text].
3.
Markus G, Evers JL, Hobika GH.
Activator activities of the transient forms of the human plasminogen-streptokinase complex during its proteolytic conversion to the stable activator complex.
J Biol Chem.
1976;251:6495-6504[Abstract/Free Full Text].
4.
Ishii K, Gerszten R, Zheng YW, Welsh JB, Turck CW, Coughlin SR.
Determinants of thrombin receptor cleavage. Receptor domains involved, specificity, and role of the P3 aspartate.
J Biol Chem.
1995;270:16435-16440[Abstract/Free Full Text].
5.
Loew D, Perrault C, Morales M, et al.
Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry.
Biochemistry.
2000;39:10812-10822[CrossRef][Medline]
[Order article via Infotrieve].
6.
Potempa J, Banbula A, Travis J.
Role of bacterial proteinases in matrix destruction and modulation of host responses.
Periodontol.
2000;24:153-192[CrossRef].
7.
Kuliopulos A, Covic L, Seeley SK, Sheridan PJ, Helin J, Costello CE.
Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy.
Biochemistry.
1999;38:4572-4585[CrossRef][Medline]
[Order article via Infotrieve].
8.
Boyle MD, Lottenberg R.
Plasminogen activation by invasive human pathogens.
Thromb Haemost.
1997;77:1-10[Medline]
[Order article via Infotrieve].
9.
McNeill H, Jensen PJ.
A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.
Cell Regul.
1990;1:843-852[Medline]
[Order article via Infotrieve].
10.
Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Coughlin SR.
PAR3 is a cofactor for PAR4 activation by thrombin.
Nature.
2000;404:609-613[CrossRef][Medline]
[Order article via Infotrieve].
Response:
Platelet-activating bacterial proteins: continuing the
emergence of a paradigm
We have read the letter by McRedmond and Fitzgerald with great
interest. We were aware of their work on
streptokinase-plasminogen-induced activation of
platelets,1 but at the time of writing our article on
gingipain activation of platelets through the PARs, the relationship of
their work to bacterial pathogenesis was not described because their
article was set in the context of the side effects experienced when
streptokinase is used in the therapeutic context of treating myocardial
infarction. The streptokinase-plasminogen-antistreptokinase antibody
complex is also obviously a far more complex interaction than that
required for gingipain activation of mammalian cells. It should also be
noted here that, as far as we are aware, the first precedent for
bacterial proteins activating mammalian cells through the PARs was in
fact our work reported in 1998,2 in which we first showed
that RgpB (which does not contain associated adhesin domains in
contrast to HrgpA) was able to activate human neutrophils through the
PAR-2 receptor. Thrombin does not activate PAR-2, and thus, combining
this with other data on the specificity of the
gingipains,3 we cannot entirely agree with the contention of McRedmond and Fitzgerald that the gingipains appear to have a
thrombinlike specificity.
McRedmond and Fitzgerald now make the case for similarities between the
action of gingipains on the PARs and that seen with the overall
streptokinase-plasminogen-antistreptokinase complex. In particular, the
authors focus upon the similar function of the associated adhesins of
HRgpA in aiding the cleavage of the PARs, much as the
streptokinase-plasminogen complex is guided to the platelet surface by
the binding of the antistreptokinase antibodies to the platelet Fc
receptor, as described in their work. Recent work in our laboratory has
shown that HRgpA and RgpB in fact have some differences in their
specificity for peptide substrates that may relate to small differences
in their active site architecture induced by a limited number of point
mutations (N. Ally et al, manuscript in preparation). We cannot
rule out that the adhesins of HrgpA play some role in enhancing
functions such as cleavage of coagulation factors and PARs, but it
would certainly appear that we can no longer be sure that it is only these adhesins that are modifying the action of the larger protein. Certainly, it must be noted that RgpB can activate the PARs directly and therefore the adhesin domains are not as vital in this process as
in the streptokinase scenario.
The letter by McRedmond and Fitzgerald now amplifies upon the role of
streptokinase-induced cellular activation in bacterial pathogenesis.
The scenario posed by them is certainly a fascinating one and, together
with our recently published work on the activation of PARs on
epithelial cells by the gingipains4 and the resulting induction of interleukin-6 secretion, the data obtained with
streptokinase certainly aids in establishing our proposal that
PAR-mediated activation of mammalian cells by bacterial proteins is an
intriguing new mechanism to be investigated in bacterial pathogenesis.
The importance of PAR-mediated platelet activation by bacterial
proteins also needs to be established further, which will provide
further intriguing glimpses into the possibility that cardiovascular
diseases might, in some cases, have an underlying chronic bacterial
disease as the cause.
Robert N. Pike, Afrodite Lourbakos, James Travis, and Jan Potempa
Correspondence: Robert N. Pike, Department of Biochemistry and
Molecular Biology, Monash University, PO Box 13D, Clayton, Victoria
3800, Australia; e-mail: rob.pike{at}med.monash.edu.au
References
1.
McRedmond JP, Harriott P, Walker B, Fitzgerald DJ.
Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1.
Blood.
2000;95:1301-1308.
2.
Lourbakos A, Chinni C, Thompson P, et al.
Cleavage and activation of protease-activated receptor-2 on human neutrophils by gingipain-R from Porphyromonas gingivalis.
FEBS Lett.
1998;435:45-48[CrossRef][Medline]
[Order article via Infotrieve].
3.
Potempa J, Mikolajczyk-Pawlinska J, Brassell D, et al.
Comparative properties of two cysteine proteases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis.
J Biol Chem.
1998;273:21648-21657[Abstract/Free Full Text].
4.
Lourbakos A, Potempa J, Travis J, et al.
An arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion.
Infect Immun.
2001;69:5121-5130[Abstract/Free Full Text].
